Enhanced stem cell therapy and stem cell production through the administration of low level light energy

ABSTRACT

Methods of enhancing stem cell therapy through the administration of low level light energy are provided in several embodiments. Some embodiments comprise irradiating stem cells before or after implantation at a target tissue having loss of function due to damage or disease, with a resultant increase in the efficacy of the cell therapy. In some embodiments, light energy enhances one or more of the viability, proliferation, migration or engraftment of the stem cells, thereby enhancing the therapeutic effects of the irradiated cells during cell therapy.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Appl. 61/229,694, filed Jul. 29, 2009 and is acontinuation-in part of U.S. patent application Ser. No. 12/817,090filed on Jun. 16, 2010, which is a continuation-in-part of U.S. patentapplication Ser. No. 11/844,205 filed on Aug. 23, 2007, which claims thebenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.60/840,370, filed Aug. 24, 2006; this application is also acontinuation-in-part of U.S. patent application Ser. No. 11/482,220,filed on Jul. 7, 2006, which is a continuation of U.S. patentapplication Ser. No. 10/682,379, filed on Oct. 9, 2003, now U.S. Pat.No. 7,303,578, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application Nos. 60/502,147, filed Sep. 11, 2003,60/487,979, filed Jul. 17, 2003, and 60/442,693, filed Jan. 24, 2003;U.S. patent application Ser. Nos. 10/682,379 and 11/482,220 each are acontinuation-in-part of U.S. patent application Ser. No. 10/287,432,filed on Nov. 1, 2002, now abandoned, which claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Application Nos. 60/369,260, filedApr. 2, 2002 and 60/336,436, filed Nov. 1, 2001; this application isalso a continuation-in-part of U.S. patent application Ser. No.12/435,274, filed May 4, 2009, which is a continuation of U.S. patentapplication Ser. No. 10/764,986, filed Jan. 26, 2004, now U.S. Pat. No.7,534,255, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application Nos. 60/537,190, filed Jan. 19, 2004,60/487,979, filed Jul. 17, 2003, and 60/442,693, filed Jan. 24, 2003,all incorporated by reference in their entireties herein.

BACKGROUND

Field of the Invention

The present application relates to systems and methods for enhancing theefficacy of various aspects of stem cell therapy. Several embodimentsare directed to enhancing one or more of the isolation, proliferation,delivery, engraftment, differentiation, or function of stem cells.Several embodiments are directed to enhancing neurologic function inindividuals having a loss of one or more neurologic functions, includingbut not limited to, motor function, cognitive function, including thatresulting from injury, neurological disorders, normal age-relateddegeneration, etc. Other embodiments are directed to improving theviability or culturability of stem cells to be used in stem cell therapyor research.

Description of the Related Art

The scope of human disease that involves loss of or damage to cells isvast and includes, but is not limited to, cancers, ocular disease,neurodegenerative disease, endocrine diseases, and cardiovasculardisease. The result of these diseases is typically some degree of lossof function of particular cells, and possibly an entire organ. This maylead to compromised quality of life, disability, or death. Injury ortrauma to these cells or organs may yield similar effects.

Cell therapy involves the use of cells, and in some cases fetal,umbilical cord, placenta-derived, adult, induced pluripotent, or humanembryonic stem cells and/or their partially or fully differentiatedcellular derivatives to treat diseased or damaged tissues viareplacement or regeneration. It is rapidly coming to the forefront oftechnologies that are poised to treat many diseases, in particular thosethat affect individuals who are non-responsive to traditionalpharmacologic therapies. In some cases, cell therapy may be used priorto, or in response to, a therapy that itself induces damage to cells ortissues.

By way of example, bone marrow contains hematopoietic stem cells (HSC),which are precursor cells not dedicated to any particular blood celllineage. Upon stimulation by particular cytokines the HSC may becomecommitted to differentiating into cells of a particular lineage.Neutrophils, which are the predominant circulating white blood cells andaccount for nearly 70% of the total white cell count (normal range of4×10⁹ to 11×10⁹ white blood cells/L of blood), are formed when HSCbecome committed to the granulocyte and/or macrophage lineage.Granulocyte colony-stimulating factor (G-CSF) is one example of amolecule that can induce the HSC to commit to forming neutrophils.

G-CSF (e.g., filgrastim, NEUPOGEN® by Amgen) is a cytokine produced byvascular endothelium and multiple types of immune cells. A G-CSFreceptor (G-CSF-R) is present on HSC in the bone marrow. Upon binding tothe G-CSF-R, G-CSF stimulates the proliferation of HSC and theirdifferentiation into mature granulocytes, such as neutrophils. G-CSF isalso a potent inducer of HSC mobilization and differentiation from thebone marrow into the bloodstream. G-CSF exists naturally, and syntheticforms have been also been developed for clinical use. Otherhematopoietic stem cell stimulator/mobilizers are also available, suchas Plerixafor (AMD3100, MOBOZIL® by Genzyme Corporation), a CXCR4alpha-chemokine receptor modulator, that functions to stimulate the HSCsfrom the bone marrow to the periphery. Synergy between Plerixafor andG-CSF is possible. Granulocyte-macrophage colony stimulating factor(GM-CSF) is another cytokine that can promote differentiation of HSCsinto neutrophils.

In normal humans, approximately one hundred billion neutrophils areproduced daily and function as the primary defense against bacterialinfections. Inactive neutrophils circulate in the blood stream with ahalf-life of about 12 hours. When activated, the circulating neutrophilsare recruited to infected or inflamed tissues where they can internalizeand kill a variety of microbes. Active neutrophils survive forapproximately 1-2 days in the tissue and serve to prevent or reduce thelikelihood of a large scale infection.

After their functional life-span has elapsed, neutrophils are typicallydestroyed by apoptosis, a sort of pre-programmed cell death. Circulatingneutrophils counts are a result of the balance of neutrophil productionand death. Neutropenia is a hematological disorder characterized by anabnormally low number of neutrophils (neutrophil granulocyte count below0.5×10⁹/litre). NNeutropenia can result from either decreased productionor accelerated destruction of neutrophils. Neutropenic individuals aremore susceptible to infections, including bacterial, fungal, andparasitic infections, with effects ranging from simple fevers tolife-threatening sepsis.

Alterations in neutrophil homeostasis may result from autoimmune orhereditary disorders, cancers, particularly those affecting the bloodcells, such as Hodgkin's disease or Non-Hodgkin lymphomas, stress, suchas from surgery or trauma, or medication, such as chemotherapeuticagents. Some medications may also have agranulocytosis as a side effect,such as, for example, antiepileptics, antithyroid drugs (carbimazole,methimazole, and propylthiouracil), antibiotics (penicillin,chloramphenicol and co-trimoxazole), cytotoxic drugs, gold, NSAIDs(indomethacin, naproxen, phenylbutazone), mebendazole, theantidepressant mirtazapine, and some antipsychotics (the atypicalantipsychotic clozapine). Some conditions may cause impaired neutrophilfunction without necessarily decreasing the quantitative number ofneutrophils. This can be attributable to certain medications, such assteroids, alcoholism, or conditions such as diabetes, end-stage liver orrenal disease, or immune disorders such as HIV.

Hodgkin's disease (HD) is a lymphoma, a hematological cancer thatoriginates from uncontrolled growth of a sub-type of white blood cellsknown as lymphocytes. Treatment for HD typically involves radiationtherapy, chemotherapy, or a combination of the two. Non-Hodgkinlymphomas (NHLs) are those lymphomas that are not classified as HD.Numerous classes of NHLs exist and they vary greatly in theiraggressiveness. Thus, therapy for NHLs is tailored to the particularclassification, but generally involves combinations of chemotherapy,immunotherapy, and radiation therapy.

In a broad sense, cancer is the rapid, uncontrolled growth of cells.Most chemotherapeutic agents act by inhibiting cell division,effectively targeting the fast-dividing cancer cells. However, there iscurrently no known cancer cell specific marker that targets thechemotherapeutic agents to cancerous cells. As a result, many normalcells, such as rapidly produced blood cells like neutrophils can also beaffected. In combination with additional damaging effects on bone marrowand the subsequent drop in white blood cell production, virtually allchemotherapeutic regimes can cause suppression of the immune system dueto neutropenia. It is therefore evident that when the chemotherapy istargeted to blood cells, as in HD and NHLs, the risk of neutropenia iseven greater.

While there is no ideal therapy for neutropenia, several approaches haveevolved to address neutropenia in the cancer treatment setting. Whendoses of chemotherapy are relatively low, the bone marrow may remainviable and marginally functional. In these cases, G-CSF and/oradministration of other agents concurrent with chemotherapy may be usedto combat neutropenia through the increased production of neutrophils.However, when higher doses of chemotherapy are needed, G-CSF may be usedprior to chemotherapy to stimulate proliferation of HSC, which can beharvested and later transplanted back into the patient. While theseapproaches have produced positive results, increasing production of HSCand neutrophils in cancer patients remain major hurdles.

SUMMARY

Several embodiments of the invention provide methods for the efficientproduction of hematopoietic stem cells and the treatment of neutropenia.Several embodiments further provide methods for isolating, mobilizing,stimulating, proliferating, or otherwise enhancing the effects of othertypes of stem cells. In one embodiment, the therapeutic effect of stemcells that are used in cell therapy is enhanced.

In several embodiments of the invention, a method for enhancing thesuitability of stem cells (e.g., neural) for use in cell therapy usinglow level light therapy (LLLT) is provided. In some embodiments, themethod comprises obtaining a population of stem cells, providing a LLLTdevice having a light emitting surface that emits light energy,delivering light energy to the stem cells, wherein the light energyincreases one or more of the viability, proliferation, differentiation,migration, or engraftment of the stem cells, thereby enhancing thesuitability of the stem cells for use in cell therapy. In oneembodiment, the invention enhances the suitability of neural stem cellsfor use in neural cell therapy, including but not limited to celltherapy targeted for brain tissue, spinal cord and other central andperipheral nervous system tissue.

In several embodiments, a method for enhancing the efficacy of stem celltherapy in a mammal using LLLT is provided. In one embodiment, themethod comprises identifying a mammal having a tissue with impairedfunction, administering one or more stem cells to the tissue, providinga LLLT device having a light emitting surface that emits light energy,and delivering light energy to the tissue, wherein the light energyenhances one or more of the viability, engraftment, proliferation,migration, or differentiation of the administered stem cells, therebyenhancing the efficacy of the stem cell therapy.

In several embodiments, a method for improving the efficiency of one ormore peripheral collections of stem cells in a mammal using LLLT and astem cell mobilizing compound is provided. In some embodiments, themethod comprises administering a stem cell stimulating or mobilizingcompound to a mammal, providing a LLLT device having a light emittingsurface that emits light energy, and delivering light energy to at leastone long bone of the mammal, thereby increasing the mobilization of stemcells from the bone marrow to the peripheral blood of the mammal. Inseveral embodiments the stem cell stimulating or mobilizing compoundworks synergistically with the light energy to stimulate bone marrowwithin the long bone, thereby increasing the mobilization of stem cellsfrom the bone marrow to the peripheral blood of the mammal and improvingthe efficiency of peripheral collection of stem cells.

In some embodiments, the light energy has a wavelength between about 350nm and 1200 nm. In some embodiments, the light energy has a wavelengthbetween about 500 nm and 1000 nm. In some embodiments, the light energyhas a wavelength between about 670 and 900 nm (e.g., 670, 700, 730, 760,790, 800, 810, 830, 850, 870, 900 nm). Depending on the target tissue,longer or shorter wavelengths are used. In one embodiment, the lightenergy has a time averaged irradiance at or within about one centimeterof the stem cells of at least about 0.01 mW/cm². In one embodiment, thelight energy has a time averaged irradiance at or within about onecentimeter of the stem cells of about 20 mW/cm² to about 60 mW/cm²(e.g., 20, 30, 40, 50, or 60 mW/cm²). In one embodiment, the lightenergy has a time averaged irradiance at or within about one centimeterof the stem cells of about 50 mW/cm². Depending on the target tissue(and the amount of overlying light energy absorbing and/or reflectingtissue), greater or lesser time averaged irradiances are used.

In several embodiments, the light energy is delivered continuously. Inseveral embodiments, the light energy is delivered in pulses. In someembodiments, the light energy is delivered in pulses at a frequencyranging from about 80 to about 120 Hz. Depending on the target tissue(and the amount of overlying light energy absorbing and/or reflectingtissue), lower or higher frequencies are used. In some embodiments, thepulsing frequency is adjusted over time to tailor the therapy to thecharacteristics of a particular patient (e.g., frequency adjustmentbased on patient responsiveness to cell therapy). In some embodiments,combinations of continuous and pulsed light parameters are used.

In some embodiments, the light energy is delivered to the stem cells invitro, while in some embodiments, the light energy is delivered to thestem cells in vivo (e.g., post administration to a cell therapysubject). In still other embodiments, light energy is administered bothin vitro and in vivo. In several embodiments, an ongoing regime of lightenergy administration is used (e.g., daily, twice daily administration,either in vitro, in vivo, or both). Many varied patterns of LLLTadministration are used, based on the specific disease or injury, andcell type being used for cell therapy.

In several embodiments, the stem cells are derived from one of a varietyof stem cell sources consisting of adult stem cells, embryonic stemcells, placenta-derived stem cells, bone marrow-derived stem cells,mesenchymal stem cells, adipose stem cells, and induced pluripotent stemcells. In some embodiments, the stem cells are differentiated to adesired lineage (e.g., neural) prior to administration to a cell therapysubject. In several embodiments, the stem cells are neural stem cells.In other embodiments, the stem cells differentiate in vivo afteradministration to a cell therapy subject. In some embodiments, in vitrodifferentiation is not complete (e.g., the cells are not terminallydifferentiated), but are lineage committed.

In several embodiments, the administered stem cells are autologous withrespect to the recipient. In other embodiments, the stem cells areallogeneic with respect to the recipient. In one embodiment, mesenchymalstem cells are used in allogeneic transplants due to the ability of thecells to modulate the immune response in the target tissue. In oneembodiment, the stem cells alter T-cell or antigen presenting cellfunction, thereby reducing immunologic rejection of transplanted cells.In one embodiment, the stem cells additionally reduce fibrosis in thetarget tissue.

In several embodiments, the stem cells are for use in cell therapy totreat a neurological disease or injury. For example, in someembodiments, the tissue with impaired function is neural tissue havingimpaired function due to degenerative neural disease. In someembodiments, the stem cells are administered to a subject for thetreatment of Parkinson's disease. In some embodiments, otherdegenerative diseases, such as dopaminergic impairment, Alzheimer's,amyotrophic lateral sclerosis, Huntington's disease, and/or dementia aretreated. In several embodiments, impaired neural function is a result ofinjury to the neurons. In one embodiment, LLLT and cell therapy are usedto treat the damage due to stroke. In one embodiment, cerebral ischemia(including focal cerebral ischemia), traumatic brain injury, and/orphysical trauma such as crush or compression injury in the CNS,including a crush or compression injury of the brain, spinal cord,nerves or retina, is treated.

In several embodiments, neutropenia is treated using LLLT and a stemcell mobilizing compound by stimulating release of stem cells to theperipheral blood for later readministration to the patient (or anotherpatient) in order to repopulate dwindling cell numbers due to disease.In some embodiments, such methods are used to treat Non-Hodgkinlymphoma, Hodgkin's disease, cancer, or a side-effect of a therapy forNon-Hodgkin lymphoma, Hodgkin's disease, or cancer.

In several embodiments, stem cells are mobilized, collected, andadministered to treat other diseases. For example, in some embodiments,mobilized mesenchymal stem cells are differentiated to a pancreaticlineage and used to recapitulate insulin secretion in a diabeticsubject. In one embodiment, LLLT is used to assist in mobilizing thecells, while in one embodiment, LLLT is used in the cell therapy itself.In still another embodiment, LLLT is used both in the mobilization andin the cell therapy aspects of treatment.

In certain embodiments, a method for improving hematopoietic stem cell(HSC) production and mobilization in a patient comprises administering atherapeutically-effective amount of a therapeutic agent configured toincrease the quantity of in the bloodstream and/or improve the functionof a particular type of cell in the body, such as, for example,granulocyte colony-stimulating factor (G-CSF) to the patient inconjunction with a therapeutically effective amount of electromagneticradiation (e.g. LLLT). In certain embodiments, at least a portion of thetherapeutically-effective amount of electromagnetic radiation is appliedconcurrently with the administration of the therapeutically-effectiveamount of G-CSF.

In some embodiments, a method for preventing neutropenia comprisesadministering a therapeutically-effective amount of G-CSF to the patientin conjunction with a therapeutically effective amount ofelectromagnetic radiation. In certain embodiments, at least a portion ofthe therapeutically-effective amount of electromagnetic radiation isapplied concurrently with the administration of thetherapeutically-effective amount of G-CSF. In certain such embodiments,the neutropenia may result from cancer or therapies to be used intreating the cancer.

In other embodiments, a method for treating neutropenia comprisesadministering a therapeutically-effective amount of G-CSF to the patientin conjunction with a therapeutically effective amount ofelectromagnetic radiation. In certain embodiments, at least a portion ofthe therapeutically-effective amount of electromagnetic radiation isapplied concurrently with the administration of thetherapeutically-effective amount of G-CSF. In certain such embodiments,the neutropenia is a result of cancer or therapies used to treat thecancer.

In some embodiments, there is provided a method for treating damage orillness in the central nervous system in a mammal or human, comprisingdelivering an effective amount of light energy to an in vitro culturecomprising progenitor cells, and implanting the cells into the centralnervous system of a mammal or human, wherein delivering an effectiveamount of light energy includes delivering light having a wavelength inthe visible to near-infrared wavelength range and a power density of atleast about 0.01 mW/cm² to the cells in culture. The progenitor cellsmay be treated with another therapeutic agent, for example apharmaceutical compound or biologic prior to implantation. Without beingbound by theory or a specific mechanism, the agent or combination ofagents may have the effect of stimulating or mobilizing progenitorcells.

In some embodiments, there is provided a method for treating damage ordegeneration in non-neural tissue, for example skeletal muscle, themethod comprising delivering an effective amount of light energy to anin vitro culture comprising progenitor cells. Delivering an effectiveamount of light energy includes delivering light having a wavelength inthe visible to near-infrared wavelength range and a power density of atleast about 0.01 mW/cm² to the cells in culture. The site ofimplantation is chosen to permit the implanted cells to regenerate thedamaged tissue, for example by directly repopulating the damaged ordegenerating tissue, or by supporting the growth or proliferation ofendogenous cells. The site of implantation may also be irradiated bylaser light having a wavelength in the visible to near-infraredwavelength range and a power density of at least about 0.01 mW/cm².

In accordance with some embodiments there are provided methods directedtoward the enhancement of neurologic function in a subject. The methodsinclude delivering a neurologic enhancing effective amount of a lightenergy having a wavelength in the visible to near-infrared wavelengthrange to at least one area of the brain of a subject. In a preferredembodiment delivering the neurologic function enhancing effective amountof light energy includes delivering a predetermined power density oflight energy through the skull to the target area of the brain and/ordelivering light energy through the skull to at least one area of thebrain of a subject, wherein the wavelength, power density and amount ofthe light energy delivered are sufficient to cause an enhancement ofneurologic functioning.

The low level light therapy methods for enhancing neurologic functionare based in part on the new and surprising discovery that power density(i.e., power per unit area) of the light energy applied to tissueappears to be a very important factor in determining the relativeefficacy of low level light therapy, and particularly with respect toenhancing the function of neurons in both healthy and diseased states.

In accordance with one embodiment there is provided a method forpreventing heat stroke in a subject. The term “preventing” in thiscontext shall be given its ordinary meaning and shall include reducingthe severity of a later heat stroke in a subject that has undergonetreatment, reducing the incidence of heat stroke in individuals who haveundergone treatment, as well as reducing the likelihood of onset heatstroke in a subject that has undergone treatment. In one embodiment, themethod comprises delivering light energy having a wavelength in thevisible to near-infrared wavelength range through the skull to at leastone area of the brain of a subject, wherein the wavelength, powerdensity and amount of the light energy delivered are sufficient toprevent, reduce the severity, or reduce the incidence of heat stroke inthe subject.

In several embodiments, the target area of the brain may be all of thebrain or a specific area of the brain including, but not limited to, anarea associated with a particular cognitive or motor function, an areaexhibiting neurodegeneration, the cortex, and/or an area that has beenaffected by trauma. The subject may have a cognitive or motor impairmentsuch as from neurodegeneration or the subject may be normal.

In accordance with another embodiment, there is provided a method ofincreasing the production of ATP by neurons to increase neurologicfunction. The method comprises irradiating neurons with light energyhaving a wavelength in the near infrared to visible portion of theelectromagnetic spectrum for at least about 1 second, where the powerdensity of the light energy at the neurons is at least about 0.01mW/cm².

In certain embodiments, a method of treating a patient having neurologicfunction affected by Parkinson's disease is provided. The methodcomprises providing a patient having neurologic function affected byParkinson's disease. The method further comprises deliveringelectromagnetic radiation noninvasively through the scalp and the skullof the patient to at least one portion of the brain of the patient. Thelight energy has a wavelength in the visible to near-infrared wavelengthrange, and the wavelength, power density (or irradiance), and amount ofthe light energy delivered to the at least one portion of the brain aresufficient to reduce the severity of symptoms of Parkinson's disease inthe patient.

In several embodiments, the predetermined power density is a powerdensity of at least about 0.01 mW/cm². The predetermined power densityin preferred embodiments is typically selected from the range of about0.01 mW/cm² to about 100 mW/cm², including from about 0.01 mW/cm² toabout 15 mW/cm² and from about 2 mW/cm² to about 50 mW/cm². In someembodiments, power densities above or below these values may be used.

In some embodiments, the methods encompass using light energy having awavelength of about 630 nm to about 904 nm, and in one embodiment thelight energy has a wavelength of about 780 nm to about 840 nm. The lightenergy is preferably from a coherent source (i.e. a laser), but lightfrom non-coherent sources may also be used.

In some embodiments, the methods encompass placing a light source incontact with a region of skin that is either adjacent an area of thebrain in which treatment is desired, contralateral to such area, or acombination of the foregoing, and then administering the light energy,including the neurologic function enhancing effective amount of lightenergy, as may be measured by power density, to the area of the brain.In delivering the light, the power density may be a predetermined powerdensity. Some preferred methods encompass determining a surface powerdensity of the light energy sufficient for the light energy to penetratethe skull. The determination of the required surface power density,which is relatively higher than the power density to be delivered to thebrain tissue being treated, takes into account factors that attenuatepower density as it travels through tissue, including skin pigmentation,and location of the brain area being treated, particularly the distanceof the brain area from the skin surface where the light energy isapplied.

In certain embodiments, a method of treating or preventing Parkinson'sdisease is provided. The method comprises noninvasively irradiating atleast a portion of a patient's brain with electromagnetic radiationtransmitted through the scalp. The electromagnetic radiation has a powerdensity (or irradiance), between 0.01 mW/cm² and 100 mW/cm² at a depthof approximately 2 centimeters below the dura.

In certain embodiments, a method of treating a patient is provided. Themethod comprises delivering electromagnetic radiation noninvasivelythrough the scalp and the skull to at least one portion of the brain ofthe patient. The light energy has a wavelength in the visible tonear-infrared wavelength range, and the wavelength, power density, andamount of the light energy delivered to the at least one portion of thebrain are sufficient to prevent, reduce the severity, or reduce theincidence of Parkinson's disease in the patient.

In certain embodiments, a method of preventing Parkinson's disease in apatient is provided. The method comprises providing a patient having apredisposition towards contracting Parkinson's disease. The methodfurther comprises delivering electromagnetic radiation noninvasivelythrough the scalp and the skull of the patient to at least one portionof the brain of the patient. The light energy has a wavelength in thevisible to near-infrared wavelength range, and the wavelength, powerdensity, and amount of the light energy delivered to the at least oneportion of the brain are sufficient to reduce a probability of thepatient contracting Parkinson's disease.

In certain embodiments, a method of treating the central nervous systemof a patient is provided. The method comprises identifying a patientexhibiting symptoms of damage to the central nervous system due toParkinson's disease. The method further comprises irradiating an invitro culture comprising progenitor cells with electromagnetic radiationhaving a wavelength in the visible to near-infrared wavelength range anda power density of at least about 0.01 mW/cm². The method furthercomprises implanting the irradiated cells into the central nervoussystem of the patient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates a therapy apparatus comprising a capwhich fits securely over the patient's head.

FIG. 2 schematically illustrates a fragmentary cross-sectional viewtaken along the lines 2-2 of FIG. 1, showing one embodiment of a portionof a therapy apparatus comprising an element and its relationship to thescalp and brain.

FIG. 3 schematically illustrates an embodiment with an elementcomprising a container coupled to an inlet conduit and an outlet conduitfor the transport of a flowing material through the element.

FIG. 4A schematically illustrates a fragmentary cross-sectional viewtaken along the lines 2-2 of FIG. 1, showing another embodiment of aportion of a therapy apparatus comprising an element with a portioncontacting the scalp and a portion spaced away from the scalp.

FIG. 4B schematically illustrates a fragmentary cross-sectional viewtaken along the lines 2-2 of FIG. 1, showing an embodiment of a portionof a therapy apparatus comprising a plurality of light sources and anelement with portions contacting the scalp and portions spaced away fromthe scalp.

FIGS. 5A and 5B schematically illustrate cross-sectional views of twoembodiments of the element in accordance with FIG. 4B taken along theline 4-4.

FIGS. 6A-6C schematically illustrate an embodiment in which the lightsources are spaced away from the scalp.

FIGS. 7A and 7B schematically illustrate the diffusive effect on thelight by the element.

FIG. 8A schematically illustrates a therapy apparatus comprising a capand a light source comprising a light blanket.

FIGS. 8B and 8C schematically illustrate two embodiments of the lightblanket.

FIG. 9 schematically illustrates a therapy apparatus comprising aflexible strap and a housing.

FIG. 10 schematically illustrates a therapy apparatus comprising ahandheld probe.

FIG. 11 is a block diagram of a control circuit comprising aprogrammable controller.

FIG. 12 schematically illustrates a therapy apparatus comprising a lightsource and a controller.

FIG. 13 schematically illustrates a light source comprising a laserdiode and a galvometer with a mirror and a plurality of motors.

FIGS. 14A and 14B schematically illustrate two irradiation patterns thatare spatially shifted relative to each other.

FIG. 15 schematically illustrates an example therapy apparatus inaccordance with embodiments described herein.

FIG. 16 schematically illustrates an example apparatus which is wearableby a patient for treating the patient's brain.

FIG. 17 schematically illustrates an example apparatus having aplurality of elements in accordance with certain embodiments describedherein.

FIG. 18 schematically illustrates an example element in an explodedview.

FIG. 19A schematically illustrates an example optical component withexample dimensions in inches.

FIGS. 19B and 19C schematically illustrate other example opticalcomponents in accordance with certain embodiments described herein.

FIG. 20 schematically illustrates an example first support ring withexample dimensions in inches.

FIG. 21 schematically illustrates an example second support ring withexample dimensions in inches.

FIG. 22 schematically illustrates an example label compatible withcertain embodiments described herein.

FIGS. 23A and 23B schematically illustrate an example labelingconfiguration for the apparatus on the left-side and right-side of theapparatus.

FIG. 23C schematically illustrates the example labeling configuration ofFIGS. 23A and 23B from above a flattened view of the apparatus.

FIGS. 24A-24E schematically illustrate various stages of structuresformed during the fabrication of the apparatus of FIGS. 17-22.

FIG. 25 schematically illustrates an apparatus which emits light forirradiating a patient's skin to treat portions of a patient's bodyunderneath the patient's skin.

FIG. 26 schematically illustrates an example optical conduit opticallycoupled to an example optical device.

FIG. 27 schematically illustrates a simplified optical device compatiblewith certain embodiments described herein.

FIG. 28A illustrates two beam profile cross-sections of a light beamemitted from the optical device of FIG. 26 with the planes of the twocross-sections of FIG. 28A generally perpendicular to one another and tothe output optical element.

FIG. 28B illustrates the encircled energy of a light beam emitted fromthe optical device of FIG. 26.

FIG. 29A illustrates two beam profile cross-sections of a light beamemitted from the optical device of FIG. 27 having a smooth gold-platedconical inner surface.

FIG. 29B illustrates the encircled energy of a light beam emitted fromthe optical device of FIG. 27.

FIG. 30 illustrates two beam profile cross-sections of a light beamemitted from the optical device of FIG. 27 having a grit sandblastedconical inner surface.

FIGS. 31A and 31B illustrate the beam divergence for the optical deviceof FIG. 26 and of FIG. 27 (with a sandblasted inner surface),respectively.

FIGS. 32A and 32B schematically illustrate two light beams havingdifferent cross-sections impinging a patient's scalp and propagatingthrough the patient's head to irradiate a portion of the patient's braintissue.

FIG. 33 is a flow diagram of an example method for controllably exposingat least one predetermined area of a patient's scalp to laser light toirradiate the patient's brain.

FIG. 34 is a schematic diagram of the electron transport chain inmitochondria.

FIG. 35 is a graph which shows mediators responsible for ischemic stroketissue damage and the time points at which they occur.

FIG. 36 is a graph of cell proliferation and cytochrome oxidase activitypercentage as functions of the wavelength of light used to stimulatemammalian cells.

FIG. 37 is a graph of the transmittance of light through blood (inarbitrary units) as a function of wavelength.

FIG. 38 is a graph of the absorption of light by brain tissue.

FIG. 39 is a graph of the efficiency of energy delivery as a function ofwavelength.

FIG. 40 is a bar graph of the absorption of 808 nanometer light throughvarious rat tissues.

FIG. 41 is a graph of the power density versus the depth from the durafor an input power density of 10 mW/cm².

FIG. 42 depicts a laser device used in accordance with severalembodiments described herein. In particular, FIG. 42 shows a hand-heldlaser device irradiating the long bone (femur) of the upper leg of apatient.

FIG. 43A is a graph of the effects of laser treatment of 7.5 mW/cm² fora treatment duration of 2 minutes on a population of rabbits havingsmall clot embolic stroke.

FIG. 43B is a graph of the effects of laser treatment of 25 mW/cm² for atreatment duration of 10 minutes on a population of rabbits having smallclot embolic stroke.

FIG. 44 is a graph showing the therapeutic window for laser-inducedbehavioral improvements after small-clot embolic strokes in rabbits.

DETAILED DESCRIPTION

As discussed above, injury and/or disease can result in the loss offunction or death of cells in a tissue afflicted with or indirectlyimpacted by the disease or injury. For example, age-related degenerationof tissues can lead to loss of function of neurons in the eye, loss oftactile sensations, reduced control over muscle movement, memoryfailure, among many other possible effects. Non-neural tissues are alsosubject to damage or disease. For example, cardiac tissue may be damagedafter an adverse myocardial event, such as a myocardial infarction orstroke. As discussed above, blood cells may be damaged by chemotherapyor radiation therapy. Liver cells may be damaged by toxins or metabolicwaste by products. These diseases and/or injuries, among others, are allcandidates for cell therapy.

Cell therapy, the introduction of new cells into a tissue in order totreat a disease, represents a possible method for repairing or replacingdiseased tissue with healthy tissue.

In several embodiments described herein, low level laser therapy, alsoreferred to as low level light therapy (“LLLT”) is used to augment theeffects of cell therapy. As described in more detail below, LLLT is usedin several embodiments to enhance the viability of stem cells. Inseveral embodiments, enhanced viability is manifest as a more robustpopulation of cells to transplant into a subject requiring cellulartherapy. In some embodiments, stem cells exposed to LLLT proliferate toa greater degree, have enhance survival post-implantation, haveincreased differentiation, and the like. In some embodiments, LLLTenhances the activation and differentiation of endogenous stem cells. Insome embodiments, LLLT is used to treat harvested stem cells (orcultured stem cells) prior to administration to an individual requiringtherapy. In some embodiments, stem cells are administered and then LLLTis employed. In some embodiments, cells are administered withoutpreviously exposing the cells the LLLT (e.g., cells not exposed untilafter administration). In some embodiments, a target tissue ispre-treated to LLLT prior to administration of cells (which either haveor have not yet been treated with LLLT). In several embodiments, cellsare treated with LLLT, then incubated for a period of time prior toadministration. For example, the incubation period ranges from a one ormore minutes to about 48 hours, in some embodiments. In someembodiments, the incubation period ranges from about 1 to about 5minutes, about 5 to about 10 minutes, about 10 to about 15, minutes,about 15 to about 20 minutes, about 20 minutes to about 30 minutes,about 30 minutes to about 40 minutes, about 40 minutes to about 50minutes, about 50 minutes to about 60 minutes, and overlapping rangesthereof. In some embodiments, the post-administration waiting period isfrom 1-4, 4-8, 8-12, 12-16, 16-20, 20-24 hours, and overlapping rangesthereof. Longer or shorter incubation periods are used in someembodiments. In some embodiments, cells are administered concurrentlywith LLLT administration to the target tissue. In some embodiments,cells are administered to a subject, a period of time elapses, and thenLLLT is used to treat both the cells and the target tissue. For example,the post-administration waiting period ranges from a one or more minutesto about 48 hours, in some embodiments. In some embodiments, thepost-administration waiting period ranges from about 1 to about 5minutes, about 5 to about 10 minutes, about 10 to about 15, minutes,about 15 to about 20 minutes, about 20 minutes to about 30 minutes,about 30 minutes to about 40 minutes, about 40 minutes to about 50minutes, about 50 minutes to about 60 minutes, and overlapping rangesthereof. In some embodiments, the post-administration waiting period isfrom 1-4, 4-8, 8-12, 12-16, 16-20, 20-24 hours, and overlapping rangesthereof. Longer or shorter incubation periods are used in someembodiments. LLLT in conjunction with stem cells can, in severalembodiments, enhance the effects of the stem cells and advantageousprovides improved therapy for a wide variety of clinical applications.

Low Level Light Therapy

High power density laser radiation is now well accepted as a surgicaltool for cutting, cauterizing, and ablating biological tissue. Highenergy lasers are now routinely used for vaporizing superficial skinlesions and, to make deep cuts. For a laser to be suitable for use as asurgical laser, it must provide laser energy at a power sufficient toheart tissue to temperatures over 50° C. Power outputs for surgicallasers vary from 1-5 W for vaporizing superficial tissue, to about 100 Wfor deep cutting.

In contrast, LLLT involves therapeutic administration of laser energy toa patient at vastly lower power outputs than those used in high energylaser applications, resulting in desirable biological (e.g.,biostimulatory) effects while leaving tissue undamaged. For example, inrat models of myocardial infarction and ischemia-reperfusion injury, lowenergy laser irradiation reduces infarct size and left ventriculardilation, and enhances angiogenesis in the myocardium. (Yaakobi et al.,J. Appl. Physiol. 90, 2411-19 (2001)). LLLT has been described fortreating pain, including headache and muscle pain, and inflammation. Asdiscussed in herein, LLLT alters one or more characteristics of stemcells (either endogenous or delivered) that yields improved therapeuticeffects in cellular therapy.

Certain embodiments described herein and related to LLLT methods forenhancing stem cell function and therapeutic benefit are based in parton the new and surprising discovery that power density (i.e., power perunit area or irradiance; as used herein, these terms areinterchangeable) of the light energy applied to tissue appears to be animportant factor in determining the relative efficacy of low level lighttherapy, and particularly with respect to enhancing the function ofneurons in both healthy and diseased states.

Several embodiments described herein provide methods directed toward theenhancement of neurologic function in a subject. In several embodiments,the methods include delivering a neurologic enhancing effective amountof a light energy having a wavelength in the visible to near-infraredwavelength range to at least one area of the brain of a subject. Incertain embodiments, delivering the neurologic function enhancingeffective amount of light energy includes delivering a predeterminedpower density of light energy through the skull to the target area ofthe brain and/or delivering light energy through the skull to at leastone area of the brain of a subject, wherein the wavelength, powerdensity and amount of the light energy delivered are sufficient to causean enhancement of neurologic functioning. As discussed herein, in otherembodiments, LLLT is delivered to other target tissues to treat diseaseor injury, and/or to potentiate the efficacy of cell therapy.

LLLT, also referred to as phototherapy or laser therapy, involvestherapeutic administration of light energy to a patient at lower poweroutputs than those used for cutting, cauterizing, or ablating biologicaltissue, which, in several embodiments, results in desirable biological(e.g., biostimulatory) effects while leaving tissue undamaged. Innon-invasive phototherapy, it is desirable, in some embodiments, toapply an efficacious amount of light energy to the internal tissue to betreated using light sources positioned outside the body.

Laser therapy has been shown to be effective in a variety of settings,including treating lymphoedema and muscular trauma, and carpal tunnelsyndrome. According to several embodiments, laser-generated infraredradiation penetrates various tissues, including the brain, and modifiesfunction. In some embodiments, laser-generated infrared radiation caninduce angiogenesis, modify growth factor (transforming growth factor-β)signaling pathways, and enhance protein synthesis.

In some embodiments, absorption of the light energy by interveningtissue can limit the amount of light energy delivered to the targettissue site, while heating the intervening tissue. In addition,scattering of the light energy by intervening tissue can limit the powerdensity or energy density delivered to the target tissue site. Bruteforce attempts to circumvent these effects by increasing the powerand/or power density applied to the outside surface of the body canresult in damage (e.g., burning) of the intervening tissue.

Non-invasive phototherapy methods according to several embodiments arecircumscribed by setting selected treatment parameters within specifiedlimits so as to preferably avoid damaging the intervening tissue. Areview of the existing scientific literature in this field would castdoubt on whether a set of undamaging, yet efficacious, parameters couldbe found. However, certain embodiments, as described herein, providedevices and methods which can achieve this goal.

Such embodiments may include selecting a wavelength of light at whichthe absorption by intervening tissue is below a damaging level. Inseveral embodiments, wavelengths of light are used at which theabsorption by intervening tissue is below a level that inhibits(partially or fully) the normal function of cells within the targettissue or the target tissue as whole. Such embodiments may also includesetting the power output of the light source at very low, yetefficacious, power densities (e.g., between approximately 100 μW/cm² toapproximately 500 μW/cm², approximately 500 μW/cm² to approximately 2.5mW/cm², approximately 2.5 mW/cm² to approximately 5 mW/cm²,approximately 5 mW/cm² to approximately 1 W/cm², approximately 1 W/cm²to approximately 5 W/cm², approximately 5 W/cm² to approximately 10W/cm², and overlapping ranges thereof)) at the target tissue site, andtime periods of application of the light energy at a few seconds tominutes to achieve an efficacious energy density at the target tissuesite being treated. Other parameters can also be varied in the use ofphototherapy. In some embodiments, these other parameters contribute tothe light energy that is actually delivered to the treated tissue andmay play key roles in the efficacy of phototherapy in augmenting theeffect of stem cell therapy.

In certain embodiments, the irradiated portion of the brain (or othertissue) can comprise the entire brain (or tissue), or portions thereof(e.g., less than 0.1%, 0.5%, 1%, 5%, 10%, 15%, 25%, 50%, or 75% of thetarget area). In one embodiment, specific cells or cell-types aretreated.

As used herein, the term “neurodegeneration” shall be given its ordinarymeaning and shall also to the process of cell destruction resulting fromprimary destructive events such as stroke or trauma, and also secondary,delayed and progressive destructive mechanisms that are invoked by cellsdue to the occurrence of the primary destructive event. Primarydestructive events include disease processes or physical injury orinsult, including stroke, but also include other diseases and conditionssuch as multiple sclerosis, amyotrophic lateral sclerosis, heat stroke,epilepsy, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, dopaminergic impairment, dementia resulting from other causessuch as AIDS, cerebral ischemia including focal cerebral ischemia, andphysical trauma such as crush or compression injury in the CNS,including a crush or compression injury of the brain, spinal cord,nerves or retina, or any other acute injury or insult producingneurodegeneration. Secondary destructive mechanisms include anymechanism that leads to the generation and release of neurotoxicmolecules, including apoptosis, depletion of cellular energy storesbecause of changes in mitochondrial membrane permeability, release orfailure in the reuptake of excessive glutamate, reperfusion injury, andactivity of cytokines and inflammation. Both primary and secondarymechanisms may contribute to forming a “zone of danger” for neurons,wherein the neurons in the zone have at least temporarily survived theprimary destructive event, but are at risk of dying due to processeshaving delayed effect.

As used herein, the term “neuroprotection” shall be given its ordinarymeaning and shall also refer to a therapeutic strategy for slowing orpreventing the otherwise irreversible loss of neurons due toneurodegeneration after a primary destructive event, whether theneurodegeneration loss is due to disease mechanisms associated with theprimary destructive event or secondary destructive mechanisms.

As used herein, the term “cognitive function” as used herein shall begiven its ordinary meaning and shall also refer to cognition andcognitive or mental processes or functions, including those relating toknowing, thinking, learning, perception, memory (including immediate,recent, or remote memory), and judging. Symptoms of loss of cognitivefunction can also include changes in personality, mood, and behavior ofthe patient. Diseases or conditions affecting cognitive function includeAlzheimer's disease, dementia, AIDS or HIV infection, Cruetzfeldt-Jakobdisease, head trauma (including single-event trauma and long-term traumasuch as multiple concussions or other traumas which may result fromathletic injury), Lewy body disease, Pick's disease, Parkinson'sdisease, Huntington's disease, drug or alcohol abuse, brain tumors,hydrocephalus, kidney or liver disease, stroke, depression, and othermental diseases which cause disruption in cognitive function, andneurodegeneration.

As used herein, the term “motor function” as used herein shall be givenits ordinary meaning and shall also refer to those bodily functionsrelating to muscular movements, primarily conscious muscular movements,including motor coordination, performance of simple and complex motoracts, and the like.

As used herein, the term “neurologic function” as used herein shall begiven its ordinary meaning and shall also refer to both cognitivefunction and motor function.

As used herein, the terms “cognitive enhancement” and “motorenhancement” as used herein shall be given its ordinary meaning andshall also refer to the improving or heightening of cognitive functionand motor function, respectively.

As used herein, the term “neurologic enhancement” as used herein shallbe given its ordinary meaning and shall also include both cognitiveenhancement and motor enhancement.

As used herein, the term “neuroprotective effective” as used hereinshall be given its ordinary meaning and shall also refer to acharacteristic of an amount of light energy, wherein the amount is apower density of the light energy measured in mW/cm². The amount oflight energy achieves the goal of preventing, avoiding, reducing oreliminating neurodegeneration, which should result in cognitiveenhancement and/or motor enhancement.

As used herein, the term “neurologic function enhancement effective” asused herein shall be given its ordinary meaning and shall also refer toa characteristic of an amount of light energy, wherein the amount is apower density of the light energy measured in mW/cm² (or anotherart-recognized unit of measure). The amount of light energy achieves thegoal of neuroprotection, motor enhancement and/or cognitive enhancement,and/or enhancement of stem cell viability, proliferation,differentiation, or increased efficacy of cell therapy.

Thus, a method for the treatment or enhancement of neurologic functionin a patient in need of such treatment involves delivering a neurologicfunction enhancement effective amount or a neuroprotective-effectiveamount of light energy having a wavelength in the visible tonear-infrared wavelength range to a target area of the patient's brain20. In certain embodiments, the target area of the patient's brain 20includes an area exhibiting neurodegeneration. In other embodiments, thetarget area includes portions of the brain 20 not exhibitingneurodegeneration. Without being bound by theory or by a specificmechanism, it is believed that irradiation of healthy tissue inproximity to the area exhibiting neurodegeneration increases theproduction of ATP and copper ions in the healthy tissue and which thenmigrate to cells exhibiting neurodegeneration, thereby producingbeneficial effects. Additional information regarding the biomedicalmechanisms or reactions involved in phototherapy is provided by Tiina I.Karu in “Mechanisms of Low-Power Laser Light Action on Cellular Level”,Proceedings of SPIE Vol. 4159 (2000), Effects of Low-Power Light onBiological Systems V, Ed. Rachel Lubart, pp. 1-17, which is incorporatedin its entirety by reference herein. In a preferred embodiment,delivering the neurologic function enhancement effective amount of lightenergy includes selecting a surface power density of the light energysufficient to deliver such predetermined power density of light energyto the target area of the brain or other tissue. Likewise, a method forpreventing, reducing the severity of a later heat stroke in a subject,reducing the incidence of future heat stroke, and/or reducing thelikelihood of onset heat stroke in a subject includes delivering lightenergy having a wavelength in the visible to near-infrared wavelengthrange and a predetermined power density through the skull to at leastone area of the brain of a subject, wherein the wavelength, powerdensity and amount of the light energy delivered are sufficient toprevent, reduce the severity, or reduce the incidence of heat stroke inthe subject.

In certain embodiments, a method treats a subject suffering fromParkinson's disease. The method includes delivering light energy havinga wavelength in the visible to near-infrared wavelength range throughthe skull to at least one target area of the brain of the subject,wherein the wavelength, power density and amount of the light energydelivered are sufficient to prevent, reduce the severity, or reduce theincidence of Parkinson's disease in the subject.

In certain embodiments, the target area of the brain may be all of thebrain or a specific area of the brain including, but not limited to, anarea associated with a particular cognitive or motor function, an areaexhibiting neurodegeneration, the cortex, and/or an area that has beenaffected by trauma. The subject may have a cognitive or motor impairmentsuch as from neurodegeneration or the subject may be normal.

In certain embodiments, the predetermined power density is a powerdensity of at least about 0.01 mW/cm². The predetermined power densityin certain embodiments is typically selected from the range of about0.01 mW/cm² to about 100 mW/cm². In certain embodiments, power densitiesabove or below these values may be used. To deliver the predeterminedpower density at the level of the brain tissue, a required, relativelygreater surface power density of the light energy is calculated takinginto account attenuation of the light energy as it travels from the skinsurface through various tissues including skin, bone and brain tissue.Factors known to affect penetration and to be taken into account in thecalculation include skin pigmentation, the presence and color of hairover the area to be treated (if any), and the location of the affectedbrain region, particularly the depth of the area to be treated relativeto the surface. For example, to obtain a desired power density of 50mW/cm² at the cortical surface of the brain may require a surface powerdensity of approximately 3500 mW/cm². When targeting depths furtherbelow the cortical surface (e.g., ˜3 cm below the surface) an increasedpower density may be required. Likewise, when targeting more superficialtissues, a lower power density is used in certain embodiments. Certaincharacteristics of the target tissue define the particular power densityrequirements. As discussed above, the scalp, blood, bone and otherintervening tissues absorb some of the administered light. With a higherlevel of skin pigmentation, a higher surface power density is requiredto deliver a predetermined power density of light energy to a subsurfacebrain site. Thus, adjustments are made in power density applieddepending on patient characteristics, target tissue depth, and theamount and content of any intervening tissues. The light energy can havea predetermined power density at the subdermal target tissue (e.g., at adepth of approximately 2 centimeters below the dura). It is presentlybelieved that phototherapy of tissue is most effective when irradiatingthe target tissue with power densities of light of at least about 0.01mW/cm² and up to about 1 W/cm². In various embodiments, the subsurfacepower density is at least about 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 15, 20,30, 40, 50, 60, 70, 80, or 90 mW/cm², respectively, depending on thedesired clinical performance. In some embodiments, the subsurface powerdensity is selected from a range of about 0.01 mW/cm² to about 15mW/cm², or of about 2 mW/cm² to about 50 mW/cm². In certain embodiments,the subsurface power density is preferably about 0.01 mW/cm² to about100 mW/cm², more preferably about 0.01 mW/cm² to about 50 mW/cm², andmost preferably about 2 mW/cm² to about 20 mW/cm². It is believed thatthese subsurface power densities are especially effective at producingthe desired biostimulative effects on the tissue being treated. However,in other embodiments, higher or lower power densities are used togenerate the desired effects on the target tissue.

In certain embodiments, the methods encompass using light energy havinga wavelength of about 630 nanometers to about 904 nanometers, and incertain embodiments the light energy has a wavelength of about 780nanometers to about 840 nanometers. In one embodiment, the light energyis preferably from a coherent source (i.e. a laser, for example a GaAIAslaser diode), but light from non-coherent sources may also be used. Insome embodiments, the light is substantially monochromatic (i.e. onewavelength or a very narrow band of wavelengths).

In certain embodiments, the methods encompass placing a light source incontact with a region of skin that is either adjacent an area of thebrain or other organ in which treatment is desired, contralateral tosuch area, or a combination of the foregoing, and then administering thelight energy, including the neurologic function enhancing effectiveamount of light energy, as may be measured by power density, to thetarget area of the brain. To treat a patient, including those sufferingfrom neurodegeneration or a loss or diminishment of motor skills,cognition or cognitive or mental processes or functions, as well aspersons having generally normal cognitive or motor functions (whether toenhance such functions or to pre-treat so as to prevent or lessen heatstroke), or to potentiate and/or otherwise improve the efficacy of celltherapy for other diseases, the light source is placed in contact with aregion of skin, for example on the scalp, adjacent a target area of thebrain. The target area may be an area of the brain affected by diseaseor trauma that has been identified such as by using standard medicalimaging techniques, it may be a portion of the brain that is known tocontrol certain functions or processes, or it may be any section of thebrain, including but not limited to the cortex, cerebellum and otherbrain regions. In delivering the light, the power density may be apredetermined power density. In certain embodiments, a surface powerdensity of the light energy sufficient for the light energy to penetratethe skull is determined. The determination of the required surface powerdensity, which is relatively higher than the power density to bedelivered to the brain (or other) tissue being treated, takes intoaccount factors that attenuate power density as it travels throughtissue, including skull thickness of the patient (or other interveningtissues), skin pigmentation, and location of the tissue being treated,particularly the distance of the brain area from the skin surface wherethe light energy is applied. The power and other parameters are thenadjusted according to the results of the calculation.

In certain embodiments, a method increases the production of adenosinetriphosphate (ATP) by neurons to increase neurologic function. Themethod comprises irradiating neurons with light energy having awavelength in the near infrared to visible portion of theelectromagnetic spectrum for at least about 1 second, where the powerdensity of said light energy at the neurons is at least about 0.01mW/cm². In other embodiments, ATP is increased in other cell types.

In several embodiments, the treatment proceeds continuously for a periodof about 30 seconds to about 2 hours, more preferably for a period ofabout 1 to 20 minutes. The treatment may be terminated after onetreatment period, or the treatment may be repeated with preferably about1 to 2 days passing between treatments. The length of treatment time andfrequency of treatment periods depends on several factors, including thefunctional recovery of the patient and the results of imaging analysis.In some cases, such as where the disease is degenerative (e.g.Alzheimer's disease) or where treatment is given to a generally healthypatient, the treatment may continue at chosen intervals indefinitely.

The precise power density selected for treating the patient will dependon a number of factors, including the specific wavelength of lightselected, the type of disease (if any), the clinical condition of thesubject including the extent of brain area affected, and the like.Similarly, it should be understood that the power density of lightenergy to be delivered to the target area or affected brain area may beadjusted to be combined with any other therapeutic agent or agents,especially pharmaceutical agents to achieve the desired biologicaleffect. The selected power density will again depend on a number offactors, including the specific light energy wavelength chosen, theindividual additional therapeutic agent or agents chosen, and theclinical condition of the subject.

During the treatment, the light energy may be continuously provided, orit may be pulsed. If the light is pulsed, the pulses are preferably atleast about 10 ns long and occur at a frequency of up to about 100 Hz.In some embodiments, light is pulsed at a frequency of up to about 100kHz. In some embodiments, pulsed light is administered at a frequencyranging from about 100 Hz to 500 Hz, 100 Hz to 1 kHz, 1 kHz to 10 kHz,10 kHz to 20 kHz, 20 kHz to 30 kHz, 30 kHz to 40 kHz, 40 kHz to 50 kHz,50 kHz to 60 kHz, 60 kHz to 70 kHz, 70 kHz to 80 kHz, 80 kHz to 90 kHz,90 kHz to 100 kHz, and overlapping ranges thereof. Continuous wave lightis also be used, in some embodiments.

It has been discovered that treatment of stroke using low level lighttherapy is more effective if treatment begins several hours after thestroke has occurred. This is a surprising result, in that thethrombolytic therapies currently in use for treatment of stroke mustbegin within a few hours of the stroke. Because oftentimes many hourspass before a person who has suffered a stroke receives medicaltreatment, the short time limit for initiating thrombolytic therapyexcludes many patients from treatment. Consequently, the present methodsmay be used to treat a greater percentage of stroke patients.Accordingly, it is believed that treatment to enhance cognitive and/ormotor function may also take place after a primary event occurs in thatit appears that the neural cells need only be living to receive benefitfrom the methods described herein.

Apparatuses for LLLT

As discussed above, phototherapy involves therapeutic administration oflight energy to a patient at lower power outputs than those used forcutting, cauterizing, or ablating biological tissue, resulting indesirable biostimulatory effects while leaving tissue undamaged. Inother embodiments, the electromagnetic radiation comprises infraredlight. In some embodiments a device may be used to administer at least aportion of the electromagnetic radiation to subdermal tissues. Incertain such embodiments, the device comprises parameters for lightadministration to a patient. Examples of devices for infrared lightadministration to a patient compatible with certain embodimentsdescribed herein are disclosed in U.S. Pat. No. 7,303,578, U.S. PatentApplication Publication Nos. 2005/0107851 A1, 2007/0179570 A1, and U.S.patent application Ser. No. 12/389,294, all of which are incorporated intheir entireties by reference herein.

Element to Inhibit Temperature Increases at an Irradiated Surface

FIGS. 1 and 2 schematically illustrate an embodiment of a therapyapparatus 10 for treating a patient's brain 20. It shall be appreciatedthat other target tissues are treated in several embodiments disclosedherein. The therapy apparatus 10 comprises a light source 40 having anoutput emission area 41 positioned to irradiate a portion of the brain20 with an efficacious power density and wavelength of light. Thetherapy apparatus 10 further comprises an element 50 interposed betweenthe light source 40 and the patient's scalp 30. The element 50 isadapted to inhibit temperature increases at the scalp 30 caused by thelight.

As used herein, the term “element” is used in its broadest sense,including, but not limited to, as a reference to a constituent ordistinct part of a composite device. In certain embodiments, the element50 is adapted to contact at least a portion of the patient's scalp 30(see e.g., FIG. 1). In certain such embodiments, the element 50 is inthermal communication with and covers at least a portion of the scalp30. In other embodiments, the element 50 is spaced away from the scalp30 and does not contact the scalp 30.

In certain embodiments, the light passes through the element 50 prior toreaching the scalp 30 such that the element 50 is in the optical path oflight propagating from the light source 40, through the scalp 30,through the bones, tissues, and fluids of the head, to the brain. Incertain embodiments, the light passes through a transmissive medium ofthe element 50, while in other embodiments, the light passes through anaperture of the element 50. As described more fully below, the element50 may be utilized with various embodiments of the therapy apparatus 10.Similar light penetration pathways occur in other embodiments that aretargeting other tissues (e.g., light passes through skin and chest wallto target cardiac tissue).

In certain embodiments, the light source 40 is disposed on the interiorsurface of a cap 60 which fits securely over the patient's head. The cap60 provides structural integrity for the therapy apparatus 10 and holdsthe light source 40 and element 50 in place. Example materials for thecap 60 include, but are not limited to, metal, plastic, or othermaterials with appropriate structural integrity. The cap 60 may includean inner lining 62 comprising a stretchable fabric or mesh material,such as Lycra or nylon. In certain embodiments, the light source 40 isadapted to be removably attached to the cap 60 in a plurality ofpositions so that the output emission area 41 of the light source 40 canbe advantageously placed in a selected position for treatment ofParkinson's disease in any portion of the brain 20. In otherembodiments, the light source 40 can be an integral portion of the cap60.

The light source 40 illustrated by FIGS. 1 and 2 comprises at least onepower conduit 64 coupled to a power source (not shown). In someembodiments, the power conduit 64 comprises an electrical conduit whichis adapted to transmit electrical signals and power to an emitter (e.g.,laser diode or light-emitting diode). In certain embodiments, the powerconduit 64 comprises an optical conduit (e.g., optical waveguide) whichtransmits optical signals and power to the output emission area 41 ofthe light source 40. In certain such embodiments, the light source 40comprises optical elements (e.g., lenses, diffusers, and/or waveguides)which transmit at least a portion of the optical power received via theoptical conduit 64. In still other embodiments, the therapy apparatus 10contains a power source (e.g., a battery) and the power conduit 64 issubstantially internal to the therapy apparatus 10.

In certain embodiments, the patient's scalp 30 comprises hair and skinwhich cover the patient's skull. In other embodiments, at least aportion of the hair is removed prior to the phototherapy treatment, sothat the therapy apparatus 10 substantially contacts the skin of thescalp 30.

In certain embodiments, the element 50 is adapted to contact thepatient's scalp 30, thereby providing an interface between the therapyapparatus 10 and the patient's scalp 30. In certain such embodiments,the element 50 is coupled to the light source 40 and in other suchembodiments, the element is also adapted to conform to the scalp 30, asschematically illustrated in FIG. 1. In this way, the element 50positions the output emission area 41 of the light source 40 relative tothe scalp 30. In certain such embodiments, the element 50 ismechanically adjustable so as to adjust the position of the light source40 relative to the scalp 30. By fitting to the scalp 30 and holding thelight source 40 in place, the element 50 inhibits temperature increasesat the scalp 30 that would otherwise result from misplacement of thelight source 40 relative to the scalp 30. In addition, in certainembodiments, the element 50 is mechanically adjustable so as to fit thetherapy apparatus 10 to the patient's scalp 30.

In certain embodiments, the element 50 provides a reusable interfacebetween the therapy apparatus 10 and the patient's scalp 30. In suchembodiments, the element 50 can be cleaned or sterilized between uses ofthe therapy apparatus, particularly between uses by different patients.In other embodiments, the element 50 provides a disposable andreplaceable interface between the therapy apparatus 10 and the patient'sscalp 30. By using pre-sterilized and pre-packaged replaceableinterfaces, certain embodiments can advantageously provide sterilizedinterfaces without undergoing cleaning or sterilization processingimmediately before use.

In certain embodiments, the element 50 comprises a container (e.g., acavity or bag) containing a material (e.g., gel or liquid). Thecontainer can be flexible and adapted to conform to the contours of thescalp 30. Other example materials contained in the container of theelement 50 include, but are not limited to, thermal exchange materialssuch as glycerol and water. The element 50 of certain embodimentssubstantially covers the entire scalp 30 of the patient, asschematically illustrated in FIG. 2. In other embodiments, the element50 only covers a localized portion of the scalp 30 in proximity to theirradiated portion of the scalp 30.

In certain embodiments, at least a portion of the element 50 is withinan optical path of the light from the light source 40 to the scalp 30.In such embodiments, the element 50 is substantially opticallytransmissive at a wavelength of the light emitted by the output emissionarea 41 of the light source 40 and is adapted to reduce back reflectionsof the light. By reducing back reflections, the element 50 increases theamount of light transmitted to the brain 20 and reduces the need to usea higher power light source 40 which may otherwise create temperatureincreases at the scalp 30. In certain such embodiments, the element 50comprises one or more optical coatings, films, layers, membranes, etc.in the optical path of the transmitted light which are adapted to reduceback reflections.

In certain such embodiments, the element 50 reduces back reflections byfitting to the scalp 30 so as to substantially reduce air gaps betweenthe scalp 30 and the element 50 in the optical path of the light. Therefractive-index mismatches between such an air gap and the element 50and/or the scalp 30 would otherwise result in at least a portion of thelight propagating from the light source 40 to the brain 20 to bereflected back towards the light source 40.

In addition, certain embodiments of the element 50 comprise a materialhaving, at a wavelength of light emitted by the light source 40, arefractive index which substantially matches the refractive index of thescalp 30 (e.g., about 1.3), thereby reducing anyindex-mismatch-generated back reflections between the element 50 and thescalp 30. Examples of materials with refractive indices compatible withembodiments described herein include, but are not limited to, glycerol,water, and silica gels. Example index-matching gels include, but are notlimited to, those available from Nye Lubricants, Inc. of Fairhaven,Mass.

In certain embodiments, the element 50 is adapted to cool the scalp 30by removing heat from the scalp 30 so as to inhibit temperatureincreases at the scalp 30. In certain such embodiments, the element 50comprises a reservoir (e.g., a chamber or a conduit) adapted to containa coolant. The coolant flows through the reservoir near the scalp 30.The scalp 30 heats the coolant, which flows away from the scalp 30,thereby removing heat from the scalp 30 by active cooling. The coolantin certain embodiments circulates between the element 50 and a heattransfer device, such as a chiller, whereby the coolant is heated by thescalp 30 and is cooled by the heat transfer device. Example materialsfor the coolant include, but are not limited to, water or air.

In certain embodiments, the element 50 comprises a container 51 (e.g., aflexible bag) coupled to an inlet conduit 52 and an outlet conduit 53,as schematically illustrated in FIG. 3. A flowing material (e.g., water,air, or glycerol) can flow into the container 51 from the inlet conduit52, absorb heat from the scalp 30, and flow out of the container 51through the outlet conduit 53. Certain such embodiments can provide amechanical fit of the container 51 to the scalp 30 and sufficientthermal coupling to prevent excessive heating of the scalp 30 by thelight. In certain embodiments, the container 51 can be disposable andreplacement containers 51 can be used for subsequent patients.

In still other embodiments, the element 50 comprises a container (e.g.,a flexible bag) containing a material which does not flow out of thecontainer but is thermally coupled to the scalp 30 so as to remove heatfrom the scalp 30 by passive cooling. Example materials include, but arenot limited to, water, glycerol, and gel. In certain such embodiments,the non-flowing material can be pre-cooled (e.g., by placement in arefrigerator) prior to the phototherapy treatment to facilitate coolingof the scalp 30.

In certain embodiments, the element 50 is adapted to apply pressure toat least a portion of the scalp 30. By applying sufficient pressure, theelement 50 can blanch the portion of the scalp 30 by forcing at leastsome blood out the optical path of the light energy. The blood removalresulting from the pressure applied by the element 50 to the scalp 30decreases the corresponding absorption of the light energy by blood inthe scalp 30. As a result, temperature increases due to absorption ofthe light energy by blood at the scalp 30 are reduced. As a furtherresult, the fraction of the light energy transmitted to the subdermaltarget tissue of the brain 20 is increased. In certain embodiments, apressure greater than two pounds per square inch is used to blanch theirradiated portion of the scalp 30, while in certain other embodiments,a pressure of at least one pound per square inch is used to blanch theirradiated portion of the scalp 30. Other ranges of pressures forblanching the irradiated portion of the scalp 30 are also compatiblewith certain embodiments described herein. The maximum pressure used toblanch the irradiated portion of the scalp 30 is limited in certainembodiments by patient comfort levels and tissue damage levels.

FIGS. 4A and 4B schematically illustrate embodiments of the element 50adapted to facilitate the blanching of the scalp 30. In thecross-sectional view of a portion of the therapy apparatus 10schematically illustrated in FIG. 4A, certain element portions 72contact the patient's scalp 30 and other element portions 74 are spacedaway from the scalp 30. The element portions 72 contacting the scalp 30provide an optical path for light to propagate from the light source 40to the scalp 30. The element portions 72 contacting the scalp 30 alsoapply pressure to the scalp 30, thereby forcing blood out from beneaththe element portion 72. FIG. 4B schematically illustrates a similar viewof an embodiment in which the light source 40 comprises a plurality oflight sources 40 a, 40 b, 40 c.

FIG. 5A schematically illustrates one embodiment of the cross-sectionalong the line 4-4 of FIG. 4B. The element portions 72 contacting thescalp 30 comprise ridges extending along one direction, and the elementportions 74 spaced away from the scalp 30 comprise troughs extendingalong the same direction. In certain embodiments, the ridges aresubstantially parallel to one another and the troughs are substantiallyparallel to one another. FIG. 5B schematically illustrates anotherembodiment of the cross-section along the line 4-4 of FIG. 4B. Theelement portions 72 contacting the scalp 30 comprise a plurality ofprojections in the form of a grid or array. More specifically, theportions 72 are rectangular and are separated by element portions 74spaced away from the scalp 30, which form troughs extending in twosubstantially perpendicular directions. The portions 72 of the element50 contacting the scalp 30 can be a substantial fraction of the totalarea of the element 50 or of the scalp 30.

FIGS. 6A-6C schematically illustrate an embodiment in which the lightsources 40 are spaced away from the scalp 30. In certain suchembodiments, the light emitted by the light sources 40 propagates fromthe light sources 40 through the scalp 30 to the brain 20 and dispersesin a direction generally parallel to the scalp 30, as shown in FIG. 6A.The light sources 40 are preferably spaced sufficiently far apart fromone another such that the light emitted from each light source 40overlaps with the light emitted from the neighboring light sources 40 atthe brain 20. FIG. 6B schematically illustrates this overlap as theoverlap of circular spots 42 at a reference depth at or below thesurface of the brain 20. FIG. 6C schematically illustrates this overlapas a graph of the power density at the reference depth of the brain 20along the line L-L of FIGS. 6A and 6B. Summing the power densities fromthe neighboring light sources 40 (shown as a dashed line in FIG. 6C)serves to provide a more uniform light distribution at the tissue to betreated. In such embodiments, the summed power density is preferablyless than a damage threshold of the brain 20 and above an efficacythreshold.

In certain embodiments, the element 50 is adapted to diffuse the lightprior to reaching the scalp 30. FIGS. 7A and 7B schematically illustratethe diffusive effect on the light by the element 50. An example energydensity profile of the light emitted by a light source 40, asillustrated by FIG. 7A, is peaked at a particular emission angle. Afterbeing diffused by the element 50, as illustrated by FIG. 7B, the energydensity profile of the light does not have a substantial peak at anyparticular emission angle, but is substantially evenly distributed amonga range of emission angles. By diffusing the light emitted by the lightsource 40, the element 50 distributes the light energy substantiallyevenly over the area to be illuminated, thereby inhibiting “hot spots”which would otherwise create temperature increases at the scalp 30. Inaddition, by diffusing the light prior to its reaching the scalp 30, theelement 50 can effectively increase the spot size of the light impingingthe scalp 30, thereby advantageously lowering the power density at thescalp 30, as described more fully below. In addition, in embodimentswith multiple light sources 40, the element 50 can diffuse the light toalter the total light output distribution to reduce inhomogeneities.

In certain embodiments, the element 50 provides sufficient diffusion ofthe light such that the power density of the light is less than amaximum tolerable level of the scalp 30 and brain 20. In certain otherembodiments, the element 50 provides sufficient diffusion of the lightsuch that the power density of the light equals a therapeutic value atthe target tissue. The element 50 can comprise example diffusersincluding, but are not limited to, holographic diffusers such as thoseavailable from Physical Optics Corp. of Torrance, Calif. and DisplayOptics P/N SN1333 from Reflexite Corp. of Avon, Conn.

FIG. 8A schematically illustrates another embodiment of the therapyapparatus 10 which comprises the cap 60 and a light source comprising alight-emitting blanket 110. FIG. 8B schematically illustrates anembodiment of the blanket 110 comprising a flexible substrate 111 (e.g.,flexible circuit board), a power conduit interface 112, and a sheetformed by optical fibers 114 positioned in a fan-like configuration.FIG. 8C schematically illustrates an embodiment of the blanket 110comprising a flexible substrate 111, a power conduit interface 112, anda sheet formed by optical fibers 114 woven into a mesh. The blanket 110is preferably positioned within the cap 60 so as to cover an area of thescalp 30 corresponding to a portion of the brain 20 to be treated.

In certain such embodiments, the power conduit interface 112 is adaptedto be coupled to an optical fiber conduit 64 which provides opticalpower to the blanket 110. The optical power interface 112 of certainembodiments comprises a beam splitter or other optical device whichdistributes the incoming optical power among the various optical fibers114. In other embodiments, the power conduit interface 112 is adapted tobe coupled to an electrical conduit which provides electrical power tothe blanket 110. In certain such embodiments, the power conduitinterface 112 comprises one or more laser diodes, the output of which isdistributed among the various optical fibers 114 of the blanket 110. Incertain other embodiments, the blanket 110 comprises anelectroluminescent sheet which responds to electrical signals from thepower conduit interface 112 by emitting light. In such embodiments, thepower conduit interface 112 comprises circuitry adapted to distributethe electrical signals to appropriate portions of the electroluminescentsheet.

The side of the blanket 110 nearer the scalp 30 is preferably providedwith a light scattering surface, such as a roughened surface to increasethe amount of light scattered out of the blanket 110 towards the scalp30. The side of the blanket 110 further from the scalp 30 is preferablycovered by a reflective coating so that light emitted away from thescalp 30 is reflected back towards the scalp 30. This configuration issimilar to configurations used for the “back illumination” ofliquid-crystal displays (LCDs). Other configurations of the blanket 110are compatible with embodiments described herein.

In certain embodiments, the light source 40 generates light which causeeye damage if viewed by an individual. In such embodiments, theapparatus 50 can be configured to provide eye protection so as to avoidviewing of the light by individuals. For example, opaque materials canbe appropriately placed to block the light from being viewed directly.In addition, interlocks can be provided so that the light source 40 isnot activated unless the apparatus 50 is in place, or other appropriatesafety measures are taken.

The phototherapy methods for the treatments described herein may bepracticed and described using, for example, a low level laser therapyapparatus such as that shown and described in U.S. Pat. Nos. 6,214,035,6,267,780, 6,273,905 and 6,290,714, which are all incorporated in theirentirety by reference herein, as are the references incorporated byreference therein.

Another suitable phototherapy apparatus in accordance with embodimentsdescribed here is illustrated in FIG. 9. The illustrated therapyapparatus 10 includes a light source 40, an element 50, and a flexiblestrap 120 adapted for securing the therapy apparatus 10 over an area ofthe patient's head, or other treatment site or external surface of thepatient above or near a treatment site. The light source 40 can bedisposed on the strap 120 itself, or in a housing 122 coupled to thestrap 120. The light source 40 preferably comprises a plurality ofdiodes 40 a, 40 b, etc. capable of emitting light energy having awavelength in the visible to near-infrared wavelength range. The element50 is adapted to be positioned between the light source 40 and thepatient's scalp 30.

It shall be appreciated that while several embodiments are described andillustrated as being directed to therapy of the brain (via the scalp) ofa patient, the parameters, concepts, materials, methods and devicesdisclosed are also capable, in other embodiments, for the efficaciousapplication of LLLT to other tissues. As such, similar variables (e.g.,heat at skin surface overlying the heart, depth of light penetration)are accounted for in such embodiments.

The therapy apparatus 10 further includes a power supply (not shown)operatively coupled to the light source 40, and a programmablecontroller 126 operatively coupled to the light source 40 and to thepower supply. The programmable controller 126 is configured to controlthe light source 40 so as to deliver a predetermined power density tothe brain tissue 20. In certain embodiments, as schematicallyillustrated in FIG. 9, the light source 40 comprises the programmablecontroller 126. In other embodiments the programmable controller 126 isa separate component of the therapy apparatus 10.

The strap is preferably fabricated from an elastomeric material to whichis secured any suitable securing means, such as mating Velcro strips,snaps, hooks, buttons, ties, or the like. Alternatively, the strap is aloop of elastomeric material sized appropriately to fit snugly over aparticular body part, such as a particular arm or leg joint, or aroundthe chest or head. The precise configuration of the strap is subjectonly to the limitation that the strap is capable of maintaining thelight energy sources in a select position relative to the particulararea of the body or tissue being treated. In any case, the light sourcesare secured to the strap so that when the strap is positioned around abody part of the patient, the light sources are positioned so that lightenergy emitted by the light sources is directed toward the skin surfaceover which the device is secured. Various strap configurations andspatial distributions of the light energy sources are contemplated sothat the device can be adapted to treat different tissues in differentareas of the body.

In some embodiments, a strap is not used and instead the light source orsources are incorporated into or attachable onto a light cap which fitssecurely over the head thereby holding the light source or sources inproximity to the patient's head for treatment. The light cap ispreferably constructed of a stretchable fabric or mesh comprisingmaterials such as Lycra or nylon. The light source or sources arepreferably removably attached to the cap so that they may be placed inthe position needed for treatment of any portion of the brain.

In the example embodiment illustrated in FIG. 9, the housing 122comprises a layer of flexible plastic or fabric that is secured to thestrap 120. In other embodiments, the housing 122 comprises a plate or anenlarged portion of the strap 120. Various strap configurations andspatial distributions of the light sources 40 are compatible withembodiments described herein so that the therapy apparatus 10 can treatselected portions of brain tissue.

In still other embodiments, the therapy apparatus 10 for delivering thelight energy includes a handheld probe 140, as schematically illustratedin FIG. 10. The probe 140 includes a light source 40 and an element 50as described herein.

FIG. 11 is a block diagram of a control circuit comprising aprogrammable controller according to embodiments described herein. Thecontrol circuit is configured to adjust the power of the light energyemitted by the light source to generate a predetermined surface powerdensity at the scalp corresponding to a predetermined energy deliveryprofile, such as a predetermined subsurface power density, to the targettissue.

In certain embodiments, the programmable controller comprises a logiccircuit, a clock or counter coupled to the logic circuit, and an userinput/interface coupled to the logic circuit. The clock of certainembodiments provides a timing signal to the logic circuit so that thelogic circuit can monitor and control timing intervals of the appliedlight. Examples of timing intervals include, but are not limited to,total treatment times, pulsewidth times for pulses of applied light, andtime intervals between pulses of applied light. In certain embodiments,the light sources can be selectively turned on and off to reduce thethermal load on the target tissue and to deliver a selected powerdensity to particular areas of the brain.

The user input/interface of certain embodiments provides signals to thelogic circuit which the logic circuit uses to control the applied light.The interface can comprise a user interface or an interface to a sensormonitoring at least one parameter of the treatment. In certain suchembodiments, the programmable controller is responsive to signals fromthe sensor to preferably adjust the treatment parameters to optimize themeasured response. The programmable controller can thus provideclosed-loop monitoring and adjustment of various treatment parameters tooptimize the phototherapy. The signals provided by the interface from auser are indicative of parameters that may include, but are not limitedto, patient characteristics (e.g., skin type, fat percentage), selectedapplied power densities, target time intervals, and power density/timingprofiles for the applied light.

In certain embodiments, the logic circuit is coupled to a light sourcedriver. The light source driver is coupled to a power supply, which incertain embodiments comprises a battery and in other embodimentscomprises an alternating current source. The light source driver is alsocoupled to the light source. The logic circuit is responsive to thesignal from the clock and to user input from the user interface totransmit a control signal to the light source driver. In response to thecontrol signal from the logic circuit, the light source driver adjustand controls the power applied to the light sources. Other controlcircuits besides the control circuit of FIG. 11 are compatible withembodiments described herein.

In certain embodiments, the logic circuit is responsive to signals froma sensor monitoring at least one parameter of the treatment to controlthe applied light. For example, certain embodiments comprise atemperature sensor thermally coupled to the scalp to provide informationregarding the temperature of the scalp to the logic circuit. In suchembodiments, the logic circuit is responsive to the information from thetemperature sensor to transmit a control signal to the light sourcedriver so as to adjust the parameters of the applied light to maintainthe scalp temperature below a predetermined level. Other embodimentsinclude example biomedical sensors including, but not limited to, ablood flow sensor, a blood gas (e.g., oxygenation) sensor, an ATPproduction sensor, or a cellular activity sensor. Such biomedicalsensors can provide real-time feedback information to the logic circuit.In certain such embodiments, the logic circuit is responsive to signalsfrom the sensors to preferably adjust the parameters of the appliedlight to optimize the measured response. The logic circuit can thusprovide closed-loop monitoring and adjustment of various parameters ofthe applied light to optimize the phototherapy.

In certain embodiments, as schematically illustrated in FIG. 12, thetherapy apparatus comprises a light source adapted to irradiate aportion of the patient's brain (or other target tissue) with anefficacious power density and wavelength of light. The therapy apparatusfurther comprises a controller for energizing said light source, so asto selectively produce a plurality of different irradiation patterns onthe patient's scalp (or other target tissue). Each of the irradiationpatterns is comprised of a least one illuminated area that is smallcompared to the target tissue, and at least one non-illuminated area.

In certain embodiments, the light source 340 includes an apparatus foradjusting the emitted light to irradiate different portions of the scalp30. In certain such embodiments, the apparatus physically moves thelight source 40 relative to the scalp 30. In other embodiments, theapparatus does not move the light source 40, but redirects the emittedlight to different portions of the scalp 30. In an example embodiment,as schematically illustrated in FIG. 13, the light source 340 comprisesa laser diode 342 and a galvometer 344, both of which are electricallycoupled to the controller 360. The galvometer 344 comprises a minor 346mounted onto an assembly 348 which is adjustable by a plurality ofmotors 350. Light emitted by the laser diode 342 is directed toward theminor 346 and is reflected to selected portions of the patient's scalp30 by selectively moving the minor 346 and selectively activating thelaser diode 342. In certain embodiments, the therapy apparatus 310comprises an element 50 adapted to inhibit temperature increases at theirradiated surface of the patient, as described herein.

FIG. 14A schematically illustrates an irradiation pattern 370 inaccordance with embodiments described herein. The irradiation pattern370 comprises at least one illuminated area 372 and at least onenon-illuminated area 374. In certain embodiments, the irradiationpattern 370 is generated by scanning the mirror 346 so that the lightimpinges the patient's scalp 30 in the illuminated area 372 but not inthe non-illuminated area 374. Certain embodiments modify the illuminatedarea 372 and the non-illuminated area 374 as a function of time.

In some embodiments, this selective irradiation can be used to reducethe thermal load on particular locations of the scalp 30 by moving thelight from one illuminated area 372 to another. For example, byirradiating the scalp 30 with the irradiation pattern 370 schematicallyillustrated in FIG. 14A, the illuminated areas 372 of the scalp 30 areheated by interaction with the light, and the non-illuminated areas 374are not heated. By subsequently irradiating the scalp 30 with thecomplementary irradiation pattern 370′ schematically illustrated in FIG.14B, the previously non-illuminated areas 374 are now illuminated areas372′, and the previously illuminated areas 372 are now non-illuminatedareas 374′. A comparison of the illuminated areas 372 of the irradiationpattern 370 of FIG. 14A with the illuminated area 372′ of theirradiation pattern 370′ of FIG. 14B shows that the illuminated areas372, 372′ do not significantly overlap one another. In this way, thethermal load at the scalp 30 due to the absorption of the light can bedistributed across the scalp 30, thereby avoiding unduly heating one ormore portions of the scalp 30.

FIG. 15 schematically illustrates another therapy apparatus 400 inaccordance with embodiments described herein. The therapy apparatus 400comprises a plurality of light sources 410 in a housing 420. Each lightsource 410 has an output emission area positioned to irradiate acorresponding portion of the brain 20 with an efficacious power densityand wavelength of light. In certain embodiments, these portions overlapsuch that the portion of the brain 20 irradiated by two or more lightsources 410 overlap one another at least in part. As described herein,the light sources 410 can be activated by a controller (not shown) inconcert or separately to produce a predetermined irradiation pattern.

The therapy apparatus 400 of FIG. 15 further comprises a cap 430interposed between the light sources 410 and the patient's scalp 30,such that light passes through the cap 430 prior to reaching the scalp30. In certain embodiments, the cap 430 is substantially opticallytransmissive at the wavelength and reduces back reflections of thelight. The cap 430 of certain embodiments fits to the scalp 30 so as tosubstantially reduce air gaps between the scalp 30 and the cap 430. Incertain embodiments, the cap 430 comprises a material having arefractive index which substantially matches a refractive index of thescalp 30. In certain embodiments, the cap 430 comprises a materialhaving a refractive index which substantially matches a refractive indexof the skin and/or hair of the scalp 30.

In the embodiment schematically illustrated by FIG. 15, the cap 430 iswearable over the patient's scalp 30. In certain such embodiments, thepatient wears the cap 430 and is in a reclining position so as to placehis head in proximity to the light sources 410. The cap 430 is adaptedto inhibit temperature increases at the scalp 30 caused by the lightfrom the light sources 410, as described herein (e.g., by cooling thescalp 30, by blanching a portion of the scalp 30, by diffusing the lightprior to reaching the scalp 30).

FIG. 16 schematically illustrates an example apparatus 500 which iswearable by a patient for treating the patient's brain. The apparatus500 comprises a body 510 and a plurality of elements 520. The body 510covers at least a portion of the patient's scalp when the apparatus 500is worn by the patient. Each element 520 has a first portion 522 whichconforms to a corresponding portion of the patient's scalp when theapparatus 500 is worn by the patient. Each element 520 has a secondportion 524 which conforms to a light source (not shown in FIG. 16)removably contacting the element. Each element 520 is substantiallytransmissive (e.g., substantially transparent or substantiallytranslucent) to light from the light source to irradiate at least aportion of the patient's brain. In certain embodiments, the light fromthe light source after being transmitted through each element 520 has apower density which penetrates the patient's cranium to deliver anefficacious amount of light to at least a portion of the patient'sbrain.

FIG. 17 schematically illustrates an example apparatus 500 having aplurality of elements 520 in accordance with certain embodimentsdescribed herein. The body 510 shown in FIG. 17 has a plurality ofapertures 512 or openings which serve as indicators of treatment sitelocations. Each element 520 is positioned at a corresponding one of theplurality of apertures 512 and serves as an optical window. In certainembodiments, the plurality of elements 520 comprises at least about 10elements 520, while in certain other embodiments, the plurality ofelements 520 comprises 20 elements 520. In certain other embodiments,the plurality of elements 520 comprises between 15 and 25 elements 520.In certain embodiments in which the light emitting apparatus 600 isconfigured to directly contact the scalp, the apertures 512 of the body510 do not contain any elements 520, but instead are indicators oftreatment site locations through which the light emitting apparatus 600is positioned for treatment.

In certain embodiments, the body 510 comprises a hood, as schematicallyillustrated by FIG. 17, while in other embodiments, the body 510comprises a cap or has another configuration which is wearable on thepatient's head and serves as a support for orienting the elements 520 onthe patient's head. In certain embodiments, the body 510 comprises astretchable material which generally conforms to the patient's scalp. Incertain embodiments, the body 510 comprises nylon-backedpolychloroprene. In certain embodiments, the body 510 is available indifferent sizes (e.g., small, medium, large) to accommodate differentsizes of heads. In certain embodiments, the apparatus 500 is disposableafter a single use to advantageously avoid spreading infection ordisease between subsequent patients.

FIG. 18 schematically illustrates an example element 520 in an explodedview. The example element 520 comprises an optical component 532, afirst support ring 534, a second support ring 536, and a label 538.Other configurations of the element 520 are also compatible with certainembodiments described herein.

In certain embodiments, the optical component 532 comprises asubstantially transmissive (e.g., substantially transparent orsubstantially translucent) bag comprising a flexible material (which canbe biocompatible). FIG. 19A schematically illustrates an example opticalcomponent 532 with example dimensions in inches. The bag of FIG. 19Acomprises an inflatable container which contains a substantiallytransmissive liquid (e.g., water) or gel. In certain embodiments, thebag has an outer diameter within a range between about 0.5 inch andabout 3 inches. For example, the bag of FIG. 19A has an outer diameterof about 1.37 inches. In certain embodiments, the bag has a volume in arange between about 2 cubic centimeters and about 50 cubic centimeters.

Both the bag and the liquid contained within the bag are substantiallytransmissive to light having wavelengths to be applied to the patient'sbrain (e.g., wavelength of approximately 810 nanometers). In certainembodiments, the liquid has a refractive index which substantiallymatches a refractive index of the patient's scalp, therebyadvantageously providing an optical match between the element 520 andthe patient's scalp. While the example optical component 532 of FIG. 19Acomprises a single bag, in certain other embodiments, the opticalcomponent 532 comprises a plurality of bags filled with a substantiallytransparent liquid.

FIGS. 19B and 19C schematically illustrate other example opticalcomponents 532 in which the bag contains a composite material. Forexample, in FIGS. 19B and 19C, the bag contains a first material 523 anda second material 525. In certain embodiments, the first material 523comprises a soft, substantially transmissive, thermally insulativematerial (e.g., gel). Example gels compatible with certain embodimentsdescribed herein include, but are not limited to, OC-431A-LVP, OCK-451,and OC-462 optical gels available from Nye Corporation of Fairhaven,Mass. In certain embodiments, the second material 525 comprises a rigid,substantially transmissive, thermally conductive material (e.g.,silica).

In certain embodiments, as schematically illustrated in FIG. 19B, thesecond material 525 comprises a plurality of balls distributed withinthe first material 523. The balls of certain embodiments have diametersless than about 2 millimeters. In certain other embodiments, asschematically illustrated in FIG. 22C, the first material 523 comprisesa first plurality of layers and the second material 525 comprises asecond plurality of layers. The first plurality of layers is stackedwith the second plurality of layers, thereby forming a stack havingalternating layers of the first material 523 and the second material525. In certain embodiments, each layer of the first plurality of layershas a thickness less than about 2 millimeters and each layer of thesecond plurality of layers has a thickness less than about 2millimeters. In certain other embodiments, each layer of the firstplurality of layers and each layer of the second plurality of layers hasa thickness less than about 0.5 millimeter. Other configurations of thefirst material 523 and the second material 525 within the opticalcomponent 532 are also compatible with certain embodiments describedherein.

The optical component 532 of certain embodiments advantageously deformsin response to pressure applied to the first portion 522 and the secondportion 524. For example, without a load being applied, the opticalcomponent 532 of FIG. 19A has a thickness of approximately 0.41 inch,but with approximately four pounds of applied pressure, the opticalcomponent 532 of FIG. 19A has a thickness of approximately 0.315 inch.The first portion 522 of the optical component 532 advantageouslydeforms to substantially conform to a portion of the patient's skull towhich the optical component 532 is pressed. For example, in certainembodiments, the first portion 522 comprises a conformable surface ofthe optical component 532. Thus, in certain such embodiments, theoptical component 532 advantageously provides an interface with thepatient's scalp which is substantially free of air gaps. The secondportion 524 of the optical component 532 advantageously deforms tosubstantially conform to a light source being pressed thereon. Forexample, in certain embodiments, the second portion 524 comprises aconformable surface of the optical component 532. Thus, in certain suchembodiments, the optical component 532 advantageously provides aninterface with the light source which is substantially free of air gaps.

In certain embodiments, the optical component 532 advantageously servesas a heat sink to inhibit temperature increases at the patient's scalpcaused by light which is transmitted through the optical component 532.In certain such embodiments, the optical component 532 has asufficiently high heat capacity to provide an effective heat sink to thepatient's scalp. For example, for a bag filled with water (which has aheat capacity of approximately 4180 joules/kilogram-K), a generallydisk-shaped bag having a diameter of approximately 32 millimeters and athickness of approximately 10 millimeters has a sufficient volume, and asufficient heat capacity, to provide an effective heat sink. Thus, incertain embodiments, each element 520 advantageously inhibitstemperature increases at the patient's scalp caused by the lighttransmitted through the element 520.

FIG. 20 schematically illustrates an example first support ring 534 withexample dimensions in inches. In certain embodiments, the first supportring 534 comprises a substantially rigid material. Examples ofcompatible materials include, but are not limited to, plastic (e.g.,acrylonitrile butadiene styrene or ABS). As illustrated in FIG. 20, thefirst support ring 534 of certain embodiments is configured to bemounted in a corresponding aperture 512 of the body 510. The examplefirst support ring 534 illustrated in FIG. 20 comprises a generally flatportion 542, an annular portion 544, and one or more protrusions 546configured to connect to the second support ring 536, described morefully below. The generally flat portion 542 has an outer diameter whichis larger than the diameter of the corresponding aperture 512 of thebody 510 and is configured to be mechanically coupled to the body 510(e.g., by adhesive). The annular portion 544 has an outer diameter whichis smaller than or equal to the diameter of the corresponding aperture512 of the body 510 and is configured to fit through the aperture 512.The one or more protrusions 546 extend generally radially from theannular portion 544 such that the overall width of the protrusions 546and the annular portion 544 is larger than the diameter of thecorresponding aperture 512 of the body 510.

FIG. 21 schematically illustrates an example second support ring 536with example dimensions in inches. In certain embodiments, the secondsupport ring 536 comprises a substantially rigid material. Examples ofcompatible materials include, but are not limited to, plastic (e.g.,acrylonitrile butadiene styrene or ABS). As illustrated in FIG. 21, thesecond support ring 536 of certain embodiments is configured to beconnected to the one or more protrusions 546 and the annular portion 544of the first support ring 534. In certain embodiments, the secondsupport ring 536 comprises one or more recesses (not shown) which areconfigured to fit with the one or more protrusions 546 of the firstsupport ring 534. In certain such embodiments, the first support ring534 and the second support ring 536 interlock together to advantageouslyhold the element 520 in place on the body 510. In certain otherembodiments, the first support ring 534 comprises one or more recessesconfigured to mate with one or more corresponding protrusions of thesecond support ring 536.

FIG. 22 schematically illustrates an example label 538 compatible withcertain embodiments described herein. The labels 538 advantageouslyprovide one or more numbers, letters, or symbols (e.g., bar codes) toeach of the elements 520 to distinguish the various elements 520 fromone another. In certain such embodiments, the labels 538 comprise avinyl material and are mechanically coupled to the second support ring536 (e.g., by adhesive) so as to be visible to users of the lighttherapy apparatus. Other types of labels 538 are also compatible withembodiments disclosed herein, including but not limited to, labels 538which are painted or etched onto an outside surface of the secondsupport ring 536.

FIGS. 23A and 23B schematically illustrate the left-side and right-sideof the apparatus 500, respectively, showing an example labelingconfiguration for the apparatus 500. FIG. 23C schematically illustratesthe example labeling configuration of FIGS. 23A and 23B from above aflattened view of the apparatus 500. The labeling convention of FIGS.23A-23C is compatible with irradiation of both halves of the patient'sbrain. Other labeling conventions are also compatible with embodimentsdescribed herein.

In certain embodiments, the labels 538 are advantageously used to guidean operator to irradiate the patient's brain at the various treatmentsites sequentially at each of the treatment sites one at a time throughthe elements 520 in a predetermined order using a light source which canbe optically coupled to sequential elements 520. For example, for thelabeling configuration of FIGS. 23A-23C, the operator can firstirradiate element “1,” followed by elements “2,” “3,” “4,” etc. tosequentially irradiate each of the twenty treatment sites one at a time.In certain such embodiments, the order of the elements 520 is selectedto advantageously reduce temperature increases which would result fromsequentially irradiating elements 520 in proximity to one another.

In certain embodiments, the labels 538 are advantageously used to keeptrack of which elements 520 have been irradiated and which elements 520are yet to be irradiated. In certain such embodiments, at least aportion of each label 538 (e.g., a pull-off tab) is configured to beremoved from the apparatus 500 when the corresponding element 520 hasbeen irradiated. In certain embodiments, the label 538 has a codesequence which the operator enters into the controller prior toirradiation so as to inform the controller of which element 520 is nextto be irradiated. In certain other embodiments, each label 538 comprisesa bar code or a radio-frequency identification device (RFID) which isreadable by a sensor electrically coupled to the controller. Thecontroller of such embodiments keeps track of which elements 520 havebeen irradiated, and in certain such embodiments, the controller onlyactuates the light source when the light source is optically coupled tothe proper element 520.

FIGS. 24A-24E schematically illustrate various stages of structuresformed during the fabrication of the apparatus 500 of FIGS. 17-22. FIG.24A schematically illustrates the body 510 mounted on a mannequin headfixture 560. The body 510 is mounted in an inside-out configuration andis shown in FIG. 24A after each of the apertures 512 has been cut in thebody 510. In each of the apertures 512, a first support ring 534 isconnected to the body 510, as shown in FIG. 24B. In certain embodiments,a layer of adhesive (e.g., CA40 Scotch-Weld™ instant adhesive availablefrom 3M Company of Saint Paul, Minn.) is applied to a surface of theflat portion 542 which is then pressed onto the body 510 with theannular portion 544 extending through the aperture 512. FIG. 24Cschematically illustrates the optical components 532 mounted on each ofthe first support rings 534. In certain embodiments, a layer of adhesive(e.g., Loctite® 3105 ultraviolet-cured adhesive available from HenkelCorporation of Rocky Hill, Conn.) is applied to a surface of the flatportion 542 which is then pressed together with a corresponding surfaceof the optical component 532 and the adhesive is cured by application ofultraviolet light. FIG. 24D schematically illustrates the body 510 afterbeing removed from the mannequin head fixture 560 and returned to anright-side-out configuration. FIG. 24E schematically illustrates theapparatus 500 after the second support rings 536 have been mounted tothe first support rings 534 and the labels 538 have been applied to thesecond support rings 536.

FIG. 25 schematically illustrates an apparatus 600 which emits light forirradiating a patient's skin to treat portions of a patient's bodyunderneath the patient's skin. The apparatus 600 comprises a source 610of light having a wavelength which is substantially transmitted by thepatient's skin. The apparatus 600 further comprises an optical conduit620 optically coupled to the source 610. The apparatus 600 furthercomprises an optical device 630 optically coupled to the optical conduit620. The optical device 630 comprises an optical diffuser 640 opticallycoupled to the optical conduit 620. The optical device 630 furthercomprises an output optical element 650 comprising a rigid andsubstantially thermally conductive material. The output optical element650 is optically coupled to the optical conduit 620 (e.g., via theoptical diffuser 640). A portion of the light transmitted through thepatient's skin irradiates at least a portion of the patient's bodyunderneath the patient's skin with an efficacious power density oflight.

In certain embodiments, the source 610 comprises a laser which emitslight having at least one wavelength in a range between about 630nanometers and about 1064 nanometers. The laser of certain otherembodiments emits light having at least one wavelength in a rangebetween about 780 nanometers and about 840 nanometers. In certainembodiments, the laser emits light having a center wavelength ofapproximately 808 nanometers. The laser of certain embodiments iscapable of generating up to approximately 6 watts of laser light and hasa numerical aperture of approximately 0.16.

FIG. 26 schematically illustrates an example optical conduit 620optically coupled to an example optical device 630. In certainembodiments, the optical conduit 620 comprises an optical fiber 622 anda protective sheath 624 around the optical fiber. The optical fiber 622of certain embodiments is a step-index optical fiber having a numericalaperture of approximately 0.22 (e.g., a 1-millimeter diameter multimodefiber). In certain embodiments, the optical conduit 620 furthercomprises an electrically conductive conduit to transmit signals betweenthe optical device 630 and the source 610 (e.g., from trigger switchesor temperature sensors within the optical device 630) and/or to provideelectrical power to the optical device 630 (e.g., for a thermoelectriccooler).

In certain embodiments, the protective sheath 624 comprises a strainrelief apparatus 625 and a SMA connector 627 which mechanically couplesto a corresponding adjustable SMA mount 631 of the optical device 630.The protective sheath 624 of certain embodiments has a plurality ofrigid segments, with each segment having a generally cylindrical tubularshape and a longitudinal axis. Each segment is articulately coupled toneighboring segments such that an angle between the longitudinal axes ofneighboring segments is limited to be less than a predetermined angle.In certain embodiments, the protective sheath 624 allows the opticalconduit 620 to be moved and to bend, but advantageously limits theradius of curvature of the bend to be sufficiently large to avoidbreaking the optical fiber 622 therein.

The example optical device 630 schematically illustrated by FIG. 26comprises an optical diffuser 640 and an output optical element 650(e.g., a lens). In certain embodiments, the output optical element 650comprises glass (e.g., BK7 glass) which is substantially opticallytransmissive at wavelengths which are substantially transmitted by skin,but is not substantially thermally conductive. In certain otherembodiments, the output optical element 650 is rigid, substantiallyoptically transmissive at wavelengths which are substantiallytransmitted by skin, and substantially thermally conductive.

In certain embodiments, the output optical element 650 has a frontsurface facing generally towards the patient's scalp and a back surfacefacing generally away from the patient's scalp. In certain embodiments,the front surface is adapted to be placed in contact with either theskin or with an intervening material in contact with the skin duringirradiation. In certain such embodiments, the thermal conductivity ofthe output optical element 650 is sufficient to allow heat to flow fromthe front surface of the output optical element 650 to a heat sink inthermal communication with the back surface of the output opticalelement 650. In certain embodiments, the output optical element 650conducts heat from the front surface to the back surface at a sufficientrate to prevent, minimize, or reduce damage to the skin or discomfort tothe patient from excessive heating of the skin due to the irradiation.

The existence of air gaps between the output optical element 650 and thescalp can create a problem in controlling the heating of the skin by theirradiation. In certain embodiments, the output optical element 650 isplaced in contact with the skin of the scalp so as to advantageouslyavoid creating air gaps between the output optical element 650 and theskin. In certain other embodiments in which an intervening material isin contact with the skin and with the output optical element 650, theoutput optical element 650 is placed in contact with the interveningmaterial so as to advantageously avoid creating air gaps between theoutput optical element 650 and the intervening material or between theintervening material and the skin.

In certain embodiments, the thermal conductivity of the output opticalelement 650 has a thermal conductivity of at least approximately 10watts/meter-K. In certain other embodiments, the thermal conductivity ofthe output optical element 650 is at least approximately 15watts/meter-K. Examples of materials for the output optical element 650in accordance with certain embodiments described herein include, but arenot limited to, sapphire which has a thermal conductivity ofapproximately 23.1 watts/meter-K, and diamond which has a thermalconductivity between approximately 895 watts/meter-K and approximately2300 watts/meter-K.

In certain embodiments, the optical diffuser 640 receives and diffuseslight 626 emitted from the optical coupler 620 to advantageouslyhomogenize the light beam prior to reaching the output optical element650. Generally, tissue optics is highly scattering, so beamnon-uniformity less than approximately 3 millimeters in size has littleimpact on the illumination of the patient's cerebral cortex. In certainembodiments, the optical diffuser 640 advantageously homogenizes thelight beam to have a non-uniformity less than approximately 3millimeters. In certain embodiments, the optical diffuser 640 has adiffusing angle of approximately one degree.

In certain embodiments, the output optical element 650 receives thediffused light 626 propagating from the optical diffuser 640 and emitsthe light 626 out of the optical device 630. In certain embodiments, theoutput optical element 650 comprises a collimating lens. In certainembodiments, the light beam emitted from the output optical element 650has a nominal diameter of approximately 30 millimeters. The perimeter ofthe light beam used to determine the diameter of the beam is defined incertain embodiments to be those points at which the intensity of thelight beam is 1/e² of the maximum intensity of the light beam. Themaximum-useful diameter of certain embodiments is limited by the size ofthe patient's head and by the heating of the patient's head by theirradiation. The minimum-useful diameter of certain embodiments islimited by heating and by the total number of treatment sites that couldbe practically implemented. For example, to cover the patient's skullwith a beam having a small beam diameter would correspondingly use alarge number of treatment sites. In certain embodiments, the time ofirradiation per treatment site can be adjusted accordingly to achieve adesired exposure dose. In certain embodiments, the beam intensityprofile has a semi-Gaussian profile, while in certain other embodiments,the beam intensity profile has a “top hat” profile.

In certain embodiments, the optical device 630 comprises an optical lenswhich receives light from the optical conduit 620 and transmits thelight to the output optical element 650. In certain such embodiments,the output optical element 650 comprises an optical diffuser. In certainembodiments, the output optical element 650 comprises both an opticallens and an optical diffuser.

In certain embodiments, the optical device 630 further comprises a heatsink 660 thermally coupled to the output optical element 650 (e.g., by athermal adhesive, such as Resinlab EP1200 available from EllsworthAdhesives of Germantown, Wis.). By having the thermally conductiveoutput optical element 650 thermally coupled to the heat sink 660,certain embodiments advantageously provide a conduit for heat conductionaway from the treatment site (e.g., the skin). In certain embodiments,the output optical element 650 is pressed against the patient's skin andtransfers heat away from the treatment site. In certain otherembodiments in which the output optical element 650 is pressed againstan element 520 which contacts the patient's skin, as described above,the element 520 advantageously provides thermal conduction between thepatient's skin and the output optical element 650.

As schematically illustrated by FIG. 26, the heat sink 660 of certainembodiments comprises a reflective inner surface 662, a first end 664,and a second end 666. The heat sink 660 is positioned so that light 626from the optical diffuser 640 is transmitted into the first end 664,through the heat sink 660, out of the second end 666, and to the outputoptical element 650. The inner surface 662 of certain embodiments issubstantially cylindrical, while for certain other embodiments, theinner surface 662 is substantially conical. In certain embodimentshaving a conical inner surface 662, the inner surface 662 at the firstend 664 has a first inner diameter and the inner surface 662 at thesecond end 666 has a second inner diameter larger than the first innerdiameter.

In certain embodiments, the heat sink 660 comprises aluminum and thereflective inner surface is gold-plated. In certain other embodiments,the reflective inner surface 662 is roughened (e.g., by gritsandblasting) to reduce specular reflections of light from the innersurface 662.

In certain embodiments, as schematically illustrated by FIG. 26, theoptical device 630 further comprises a housing 670 comprising aplurality of ventilation slots 672. The ventilation slots 672 of certainembodiments allow air flow to remove heat from the heat sink 660,thereby cooling the heat sink 660.

In certain embodiments, the housing 670 is sized to be easily held inone hand (e.g., having a length of approximately 5½ inches). The housing670 of certain embodiments further comprises one or more protectivebumpers 674 comprising a shock-dampening material (e.g., rubber). Thehousing 670 of certain embodiments is configured so that the opticaldevice 630 can be held in position and sequentially moved by hand toirradiate selected portions of the patient's skin.

In certain embodiments, as schematically illustrated by FIG. 26, theoptical device 630 further comprises at least one trigger switch 680.The trigger switch 680 is electrically coupled to the source 610. Thetrigger switch 680 of certain embodiments is actuated by pressing theoutput optical element 650 against a surface. The source 610 of certainembodiments is responsive to the trigger switch 680 by emitting lightonly when the trigger switch 680 is actuated. Therefore, in certain suchembodiments, to utilize the optical device 630, the output opticalelement 650 is pressed against the patient's skin or against an element520, such as described above.

In certain embodiments, the optical device 630 further comprises athermoelectric cooler 690 thermally coupled to the output opticalelement 650, as schematically illustrated by FIG. 26. The thermoelectriccooler 690 of certain embodiments has a cool side thermally coupled tothe output optical element 650 and a hot side which is thermally coupledto the heat sink 660. The thermoelectric cooler 690 of certainembodiments advantageously removes heat from the output optical element650. Certain embodiments of the optical device 630 comprising athermoelectric cooler 690 which actively cools the patient's skinthereby advantageously avoiding large temperature gradients at thepatient's skin which would otherwise cause discomfort to the patient. Incertain embodiments, the optical device 630 further comprises one ormore temperature sensors (e.g., thermocouples, thermistors) whichgenerate electrical signals indicative of the temperature of the outputoptical element 650 or other portions of the optical device 630.

FIG. 27 schematically illustrates a simplified optical device 630compatible with certain embodiments described herein. The optical device630 of FIG. 27 has a smaller heat sink 660 and does not have athermoelectric cooler. As schematically illustrated by FIG. 30, the heatsink 660 of certain embodiments comprises a reflective conical innersurface 662 having a first end 664 with a first inner diameter and asecond end 666 with a second inner diameter larger than the first innerdiameter. In certain embodiments, the optical device 630 of FIG. 27 isadvantageously smaller, lighter, and more easily maneuvered by hand thanthe optical device 630 of FIG. 26.

FIG. 28A illustrates two beam profile cross-sections of a light beamemitted from the optical device 630 of FIG. 26 with the planes of thetwo cross-sections of FIG. 28A generally perpendicular to one anotherand to an output optical element 650 comprising a lens. The beamdiameter of FIG. 28A is approximately 30 millimeters. FIG. 28Billustrates the encircled energy of a light beam emitted from theoptical device 630 of FIG. 26. Approximately 90% of the encircled energyfalls within a diameter of approximately 25.7 millimeters.

FIG. 29A illustrates two beam profile cross-sections of a light beamemitted from the optical device 630 of FIG. 27 having a smoothgold-plated conical inner surface 662. The planes of the twocross-sections of FIG. 29A are generally perpendicular to one anotherand to the output optical element 650. The beam diameter of FIG. 29A isapproximately 30 millimeters. The light beam has a high flux region nearthe center of the beam profile. This high flux region qualifies as a hotspot, where a hot spot is defined as regions of the light beam in whichthe local flux, averaged over a 3 millimeter by 3 millimeter area, ismore than 10% larger than the average flux. FIG. 29B illustrates theencircled energy of a light beam emitted from the optical device 630 ofFIG. 27. Approximately 90% of the encircled energy falls within adiameter of approximately 25.6 millimeters.

In certain embodiments having a smooth inner surface 662, multiplereflections of light emitted from the optical fiber 622 at large enoughangles are focused near the output optical element 650, contributing tothe hot spot region of the beam profile. FIG. 30 illustrates two beamprofile cross-sections of a light beam emitted from the optical device630 of FIG. 27 having a grit sandblasted conical inner surface 662. Thisinner surface 662 is roughened to reduce the amount of specularreflections from the inner surface 662. In certain such embodiments, thebeam profile does not have a hot spot region. Certain embodiments of theoptical device 630 advantageously generate a light beam substantiallywithout hot spots, thereby avoiding large temperature gradients at thepatient's skin which would otherwise cause discomfort to the patient.

In certain embodiments, the beam divergence emitted from the outputoptical element 650 is significantly less than the scattering angle oflight inside the body tissue being irradiated, which is typicallyseveral degrees. FIGS. 31A and 31B illustrate the beam divergence forthe optical device 630 of FIG. 26 and of FIG. 27 (with the sandblastedinner surface 622), respectively. The beam divergence was measured bymeasuring the beam profile at two separate planes and comparing theincrease in beam diameter (e.g., the diameter that encircled 90% of theenergy) further from the output optical element 650. In certainembodiments, the beam divergence has a full angle of about 12 degrees.The numerical aperture of the optical device 630 of FIG. 26 isapproximately 0.152 and the numerical aperture of the optical device 630of FIG. 27 is approximately 0.134, which equates to a difference of lessthan approximately 2.5 degrees.

Light Parameters

It is desirable to apply an efficacious amount of light energy to theinternal tissue to be treated using light sources positioned outside thebody, as schematically illustrated in the Figures discussed herein.Examples of use of external light energy to treat internal tissues aredisclosed in U.S. Pat. Nos. 6,537,304 and 6,918,922, both of which areincorporated in their entireties by reference herein.

The various parameters of the light beam emitted from the emissionsurface of the light source are advantageously selected to providetreatment while controlling, inhibiting, preventing, minimizing, orreducing injury or discomfort to the patient due to heating of the skin,tissue, or bone by the light. While discussed separately, these variousparameters below can be combined with one another within the disclosedvalues in accordance with embodiments described herein.

Wavelength

The following section discusses theories and potential actionmechanisms, as they presently appear to the inventors, regarding theselection of wavelengths for certain embodiments of phototherapydescribed herein. The scope of the claims of the present application isnot to be construed to depend on the accuracy, relevance, or specificsof any of these theories or potential action mechanisms. Thus the claimsof the present application are to be construed without being bound bytheory or by a specific mechanism.

In certain embodiments, non-invasive delivery and heating by theelectromagnetic radiation place practical limits on the ranges ofelectromagnetic radiation wavelengths to be used in the treatment of thepatient's brain. In certain embodiments, the wavelength ofelectromagnetic radiation used in the treatment of the patient's brainis selected in view of one or more of the following considerations: (1)the ability to stimulate mitochondrial function in vitro; (2) theability to penetrate tissue; (3) the absorption in the target tissue;(4) the efficacy in ischemia models in vivo; (5) the availability oflaser sources with the desired power at the desired wavelength orwavelengths; and (6) the ability to stimulate beneficial responses incells used in cell therapy (e.g., stem cells) or the tissues to whichthose cells are delivered. The combination of these effects offers fewwavelengths to be used as a therapeutic agent in vivo. These factors canbe combined in certain embodiments to create an efficiency factor foreach wavelength. Wavelengths around 800 nanometers are particularlyefficient. In addition, 808-nanometer light has previously been found tostimulate mitochondrial function and to work in the myocardialinfarction models in rat and dog. The following discussion deals withthese considerations in more detail.

In certain embodiments, the light source 40 generates light which issubstantially monochromatic (i.e., light having one wavelength, or lighthaving a narrow band of wavelengths). So that the amount of lighttransmitted to the brain is maximized, the wavelength of the light isselected in certain embodiments to be at or near a transmission peak (orat or near an absorption minimum) for the intervening tissue. In certainsuch embodiments, the wavelength corresponds to a peak in thetransmission spectrum of tissue at about 820 nanometers. In otherembodiments, the wavelength of the light is preferably between about 630nanometers and about 1064 nanometers, more preferably between about 780nanometers and about 840 nanometers, and most preferably includeswavelengths of about 785, 790, 795, 800, 805, 810, 815, 820, 825, or 830nanometers. An intermediate wavelength in a range between approximately730 nanometers and approximately 750 nanometers (e.g., about 739nanometers) appears to be suitable for penetrating the skull, althoughother wavelengths are also suitable and may be used. In severalembodiments, shorter wavelengths are preferred, such as 600 to 700 nm,including 610, 620, 630, 640, 650, 660, 670, 680, 690, and 700 nm.

In certain embodiments, light in the visible to near-infrared wavelengthrange is used to irradiate the long bones (FIG. 46). In certainembodiments, the light has a wavelength distribution peaked at a peakwavelength and has a linewidth less than ±10 nanometers from the peakwavelength. In certain such embodiments, the light has a linewidth lessthan 4 nanometers, full width at 90% of energy. In certain embodiments,the center wavelength is (808±10) nanometers with a spectral linewidthless than 4 nanometers, full width at 90% of energy. Longer or shorterwavelengths are used in other embodiments.

In other embodiments, the light source 40 generates light having aplurality of wavelengths (e.g. applied concurrently or sequentially).For example, in certain embodiments, a band of wavelengths of (808±5)nanometers is used. In certain embodiments, the light source 40 isadapted to generate light having a first wavelength concurrently withlight having a second wavelength. In certain other embodiments, thelight source 40 is adapted to generate light having a first wavelengthsequentially with light having a second wavelength.

In certain embodiments, a single light source 40 is used as a lightgenerator to generate light, while in other embodiments, a plurality oflight sources 40 are used as a light generator to generate light. Thelight source 40 preferably generates light in the visible tonear-infrared wavelength range. In certain embodiments, the light source40 comprises one or more laser diodes, which each provide coherentlight. In embodiments in which the light from the light source 40 iscoherent, the emitted light may produce “speckling” due to coherentinterference of the light. This speckling comprises intensity spikeswhich are created by constructive interference and can occur inproximity to the target tissue being treated. For example, while theaverage power density may be approximately 10 mW/cm², the power densityof one such intensity spike in proximity to the brain (or other) tissueto be treated may be approximately 300 mW/cm². In certain embodiments,this increased power density due to speckling can improve the efficacyof treatments using coherent light over those using incoherent light forillumination of deeper tissues.

In certain embodiments, the light source 40 includes at least onecontinuously emitting GaAlAs laser diode having a wavelength of about830 nanometers. In another embodiment, the light source 40 comprises alaser source having a wavelength of about 808 nanometers. In still otherembodiments, the light source 40 includes at least one vertical cavitysurface-emitting laser (VCSEL) diode. Other light sources 40 compatiblewith embodiments described herein include, but are not limited to,light-emitting diodes (LEDs) and filtered lamps.

In certain embodiments, each wavelength is selected so as to work (e.g.,cooperate functionally) with one or more chromophores within the targettissue. In several embodiments, irradiation of chromophores increasesthe production of ATP in the target tissue and/or controls, inhibits,prevents, minimizes, or reduces apoptosis of the injured tissues,thereby producing beneficial effects, as described more fully below.

Some chromophores, such as water or hemoglobin, are ubiquitous andabsorb light to such a degree that little or no penetration of lightenergy into a tissue occurs. For example, water absorbs light aboveapproximately 1300 nanometers. Thus energy in this range has littleability to penetrate tissue due to the water content. However, water istransparent or nearly transparent in wavelengths between 300 and 1300nanometers. Another example is hemoglobin, which absorbs heavily in theregion between 300 and 670 nanometers, but is reasonably transparentabove 670 nanometers.

Based on these broad assumptions, one can define an “IR window” into thebody. Within the window, there are certain wavelengths that are more orless likely to penetrate. This discussion does not include wavelengthdependent scattering effects of intervening tissues.

The absorption/transmittance of various tissues have been directlymeasured to determine the utility of various wavelengths. For example,blood absorbs less in the region above 700 nanometers, and isparticularly transparent at wavelengths above 780 nanometers.Wavelengths below 700 nanometers are heavily absorbed, and are notlikely to be useful therapeutically (except for topical indications).However, in certain embodiments, wavelengths below 700 nm arebeneficial, for example in treating stem cells prior to delivery to asubject (e.g., as an in vitro pre-treatment).

Absorption by the target tissue can be strong in a range of wavelengths(e.g., between 620 and 980 nanometers) at which copper centers inmitochondria absorb. Thus, absorption in the range of wavelengths isexpected upon a photostimulative effect taking place.

By combining the transmittance through intervening tissue with theabsorption by target tissue, the efficiency of energy delivery as afunction of wavelength can be calculated.

Wavelengths between 780 and 880 nanometers are preferable (efficiency of0.6 or greater) for certain embodiments described herein. The peakefficiency is about 800 to 830 nanometers (efficiency of 1.0 orgreater). These wavelengths are not absorbed by water or hemoglobin, andare likely to penetrate to the long bones. Once these wavelengths reachthe long bones, they will be absorbed by the cells within the long bonesand converted to useful energy.

Power Density

Phototherapy for the treatment of neurologic conditions (e.g.,neurodegenerative diseases such as Parkinson's disease), for theenhancement of stem cell therapy, and/or for improving the viability ofstem cells (among other applications), is based in part on the conceptthat power density (i.e., power per unit area or number of photons perunit area per unit time) and energy density (i.e., energy per unit areaor number of photons per unit area) of the light energy applied totissue appear to be significant factors in determining the relativeefficacy of low level phototherapy. Certain embodiments described hereinare based at least in part on the concept that, given a selectedwavelength of light energy, it is the power density and/or the energydensity of the light delivered to tissue (as opposed to the total poweror total energy delivered to the tissue) that are important factors indetermining the relative efficacy of phototherapy.

The significance of the power density used in phototherapy hasramifications with regard to the devices and methods used inphototherapy of brain tissue, as schematically illustrated by FIGS. 8Aand 8B, which show the effects of scattering by intervening tissue.Further information regarding the scattering of light by tissue isprovided by V. Tuchin in “Tissue Optics: Light Scattering Methods andInstruments for Medical Diagnosis,” SPIE Press (2000), Bellingham,Wash., pp. 3-11, which is incorporated in its entirety by referenceherein.

FIG. 32A schematically illustrates a light beam 80 impinging a portion90 of a patient's scalp 30 and propagating through the patient's head toirradiate a portion 100 of the patient's brain tissue 20. In the exampleembodiment of FIG. 32A, the light beam 80 impinging the scalp 30 iscollimated and has a circular cross-section with a radius of 2 cm and across-sectional area of approximately 12.5 cm². For comparison purposes,FIG. 32B schematically illustrates a light beam 82 having asignificantly smaller cross-section impinging a smaller portion 92 ofthe scalp 30 to irradiate a portion 102 of the brain tissue 20. Thelight beam 82 impinging the scalp 30 in FIG. 32B is collimated and has acircular cross-section with a radius of 1 cm and a cross-sectional areaof approximately 3.1 cm². The collimations, cross-sections, and radii ofthe light beams 80, 82 illustrated in FIGS. 32A and 32B are examples;other light beams with other parameters are also compatible withembodiments described herein. In particular, similar considerationsapply to focused beams or diverging beams, as they are similarlyscattered by the intervening tissue.

As shown in FIGS. 32A and 32B, the cross-sections of the light beams 80,82 become larger while propagating through the head due to scatteringfrom interactions with tissue of the head. Assuming that the angle ofdispersion is 15 degrees and the irradiated brain tissue 20 is 2.5 cmbelow the scalp 30, the resulting area of the portion 100 of braintissue 20 irradiated by the light beam 80 in FIG. 32A is approximately22.4 cm². Similarly, the resulting area of the portion 102 of braintissue 20 irradiated by the light beam 82 in FIG. 32B is approximately8.8 cm².

Irradiating the portion 100 of the brain tissue 20 with a power densityof 10 mW/cm² corresponds to a total power within the portion 100 ofapproximately 224 mW (10 mW/cm²×22.4 cm²). Assuming only approximately5% of the light beam 80 is transmitted between the scalp 30 and thebrain tissue 20, the incident light beam 80 at the scalp 30 will have atotal power of approximately 4480 mW (224 mW/0.05) and a power densityof approximately 358 mW/cm² (4480 mW/12.5 cm²). Similarly, irradiatingthe portion 102 of the brain tissue 20 with a power density of 10 mW/cm²corresponds to a total power within the portion 102 of approximately 88mW (10 mW/cm²×8.8 cm²), and with the same 5% transmittance, the incidentlight beam 82 at the scalp 30 will have a total power of approximately1760 mW (88 mW/0.05) and a power density of approximately 568 mW/cm²(1760 mW/3.1 cm²). These calculations are summarized in Table 1.

TABLE 1 2 cm Spot Size 1 cm Spot Size (FIG. 32A) (FIG. 32B) Scalp: Area12.5 cm² 3.1 cm² Total power 4480 mW 1760 mW Power density 358 mW/cm²568 mW/cm² Brain: Area 22.4 cm² 8.8 cm² Total power 224 mW 88 mW Powerdensity 10 mW/cm² 10 mW/cm²

These example calculations illustrate that to obtain a desired powerdensity at the brain 20, higher total power at the scalp 30 can be usedin conjunction with a larger spot size at the scalp 30. Thus, byincreasing the spot size at the scalp 30, a desired power density at thebrain 20 can be achieved with lower power densities at the scalp 30which can reduce the possibility of overheating the scalp 30. In certainembodiments, the light can be directed through an aperture to define theillumination of the scalp 30 to a selected smaller area. In severalembodiments, similar calculations generate light parameters that areused to treat other tissues, such as other neural tissue (e.g., spinalcord or peripheral nerves), cardiac tissue, etc.

In certain embodiments the light energy has a time averaged irradianceor power density at the emission surface of the light source betweenabout 10 mW/cm² to about 10 W/cm², between about 100 mW/cm² to about1000 mW/cm², between about 500 mW/cm² to about 1 W/cm², or between about650 mW/cm² to about 750 mW/cm² across the cross-sectional area of thelight beam. For a pulsed light beam, the time-averaged irradiance isaveraged over a time period long compared to the temporal pulse widthsof the pulses (e.g., averaged over a fraction of a second longer thanthe temporal pulse width, over 1 second, or over multiple seconds). Fora continuous-wave (CW) light beam with time-varying irradiance, thetime-averaged irradiance can be an average of the instantaneousirradiance averaged over a time period longer than a characteristic timeperiod of fluctuations of the light beam. In certain embodiments, a dutycycle in a range between 1% and 80%, between 10% and 30%, or about 20%can be used with a peak irradiance at the emission surface 22 of theoutput optical assembly 20 between about 12.5 mW/cm² to about 1000W/cm², between about 50 mW/cm² to about 50 W/cm², between about 500mW/cm² to about 5000 mW/cm², between about 2500 mW/cm² to about 5 W/cm²,or between about 3.25 W/cm² to about 3.75 W/cm² across thecross-sectional area of the light beam. In certain embodiments, thepulsed light beam has an energy or fluence (e.g., peak irradiancemultiplied by the temporal pulsewidth) at the emission surface of thelight source between about 12.5 μJ/cm² to about 1 J/cm², between about50 μJ/cm² to about 50 mJ/cm², between about 500 μJ/cm² to about 5mJ/cm², between about 2.5 mJ/cm² to about 5 mJ/cm², or between about3.25 mJ/cm² to about 3.75 mJ/cm².

The cross-sectional area of the light beam of certain embodiments (e.g.,multimode beams) can be approximated using an approximation of the beamintensity distribution. For example, measurements of the beam intensitydistribution can be approximated by a Gaussian (1/e² measurements) or bya “top hat” distribution and a selected perimeter of the beam intensitydistribution can be used to define a bound of the area of the lightbeam. In certain embodiments, the irradiance at the emission surface ofthe light source is selected to provide the desired irradiances at thesubdermal target tissue. The irradiance of the light beam is preferablycontrollably variable so that the emitted light energy can be adjustedto provide a selected irradiance at the subdermal tissue beingirradiated. In certain embodiments, the light beam emitted from theemission surface of the light source is continuous with a total radiantpower in a range of about 4 Watts to about 6 Watts. In certainembodiments, the radiant power of the light beam is 5 Watts±20% (CW). Incertain embodiments, the peak power for pulsed light is in a range ofabout 10 Watts to about 30 Watts (e.g., 20 Watts). In certainembodiments, the peak power for pulsed light multiplied by the dutycycle of the pulsed light yields an average radiant power in a range ofabout 4 Watts to about 6 Watts (e.g., 5 Watts).

In certain embodiments, the light source 40 is capable of emitting lightenergy at a power sufficient to achieve a predetermined power density atthe subdermal target tissue (e.g., at a depth of approximately 2centimeters from the dura, at the depth of the marrow cavity of theirradiated long bones; at the myocardium, etc.). It is presentlybelieved that phototherapy of tissue is most effective when irradiatingthe target tissue with power densities of light of at least about 0.01mW/cm² and up to about 1 W/cm² at the level of the tissue. In variousembodiments, the subsurface power density is at least about 0.01, 0.05,0.1, 0.5, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, or 90 mW/cm²,respectively, depending on the desired clinical performance. In certainembodiments, the subsurface power density at the target tissue is about0.01 mW/cm² to about 100 mW/cm², about 0.01 mW/cm² to about 50 mW/cm²,about 2 mW/cm² to about 20 mW/cm², or about 5 mW/cm² to about 25 mW/cm².It is believed that these subsurface power densities are especiallyeffective at producing the desired biostimulative effects on the tissuebeing treated. In certain embodiments, a duty cycle in a range between1% and 80%, between 10% and 30%, or about 20% can be used with a peakirradiance at the target tissue of 0.05 mW/cm² to about 500 mW/cm²,about 0.05 mW/cm² to about 250 mW/cm², about 10 mW/cm² to about 100mW/cm², or about 25 mW/cm² to about 125 mW/cm².

In certain embodiments, the irradiance of the light beam is selected toprovide a predetermined irradiance at the subdermal target tissue (e.g.,the brain, the myocardium, the marrow cavity of the irradiated longbones). The selection of the appropriate irradiance of the light beamemitted from the emission surface of the light source to use to achievea desired subdermal irradiance preferably includes consideration ofscattering by intervening tissue. Further information regarding thescattering of light by tissue is provided by U.S. Pat. No. 7,303,578,which is incorporated in its entirety by reference herein, and V. Tuchinin “Tissue Optics: Light Scattering Methods and Instruments for MedicalDiagnosis,” SPIE Press (2000), Bellingham, Wash., pp. 3-11, which isincorporated in its entirety by reference herein.

Taking into account the attenuation of energy as it propagates from theskin surface, through body tissue, bone, and fluids, to the subdermaltarget tissue, surface power densities preferably between about 10mW/cm² to about 10 W/cm², or more preferably between about 100 mW/cm² toabout 500 mW/cm², will typically be used to attain the selected powerdensities at the subdermal target tissue. To achieve such surface powerdensities, the light source 40 is preferably capable of emitting lightenergy having a total power output of at least about 25 mW to about 100W. In various embodiments, the total power output is limited to be nomore than about 30, 50, 75, 100, 150, 200, 250, 300, 400, or 500 mW,respectively. In certain embodiments, the light source 40 comprises aplurality of sources used in combination to provide the total poweroutput. The actual power output of the light source 40 is preferablycontrollably variable. In this way, the power of the light energyemitted can be adjusted in accordance with a selected power density atthe subdermal tissue being treated.

Certain embodiments utilize a light source 40 that includes only asingle laser diode that is capable of providing about 25 mW to about 100W of total power output at the skin surface. In certain suchembodiments, the laser diode can be optically coupled to the scalp 30via an optical fiber or can be configured to provide a sufficientlylarge spot size to avoid power densities which would burn or otherwisedamage the scalp 30. In other embodiments, the light source 40 utilizesa plurality of sources (e.g., laser diodes) arranged in a grid or arraythat together are capable of providing at least about 25 mW to about 100W of total power output at the skin surface. The light source 40 ofother embodiments may also comprise sources having power capacitiesoutside of these limits.

Temporal Pulsewidth, Temporal Pulseshape, Duty Cycle, Repetition Rate,and Irradiance per Pulse

In some embodiments, a pulsed light beam is used having a temporalprofile comprising a plurality of pulses (P₁, P₂, . . . , P_(i)), eachpulse having a temporal pulsewidth during which the instantaneousintensity or irradiance I(t) of the pulse is substantially non-zero. Forexample, a pulse P₁ has a temporal pulsewidth from time t=0 to timet=T₁, pulse P₂ has a temporal pulsewidth from time t=T₂ to time t=T₃,and pulse P_(i) has a temporal pulsewidth from time t=T_(i) to timet=T_(i+1). The temporal pulsewidth can also be referred to as the “pulseON time.” The pulses are temporally spaced from one another by periodsof time during which the intensity or irradiance of the beam issubstantially zero. For example, pulse P₁ is spaced in time from pulseP₂ by a time t=T₂−T₁. The time between pulses can also be referred to asthe “pulse OFF time.” In certain embodiments, the pulse ON times of thepulses are substantially equal to one another, while in certain otherembodiments, the pulse ON times differ from one another. In certainembodiments, the pulse OFF times between the pulses are substantiallyequal to one another, while in certain other embodiments, the pulse OFFtimes between the pulses differ from one another. As used herein, theterm “duty cycle” has its broadest reasonable interpretation, includingbut not limited to, the pulse ON time divided by the sum of the pulse ONtime and the pulse OFF time. For a pulsed light beam, the duty cycle isless than one. The values of the duty cycle and the temporal pulsewidthfully define the repetition rate of the pulsed light beam. Furtherdisclosure regarding parameters of pulsed light compatible with certainembodiments described herein may be found in United States PatentPublication No. 2009/0254154, which is incorporated in its entirety byreference herein.

Each of the pulses can have a temporal pulseshape which describes theinstantaneous intensity or irradiance of the pulse I(t) as a function oftime. For example, the temporal pulseshapes of the pulsed light beam maybe irregular, and need not be the same among the various pulses. Incertain embodiments, the temporal pulseshapes of the pulsed light beamare substantially the same among the various pulses. For example, thepulses can have a square temporal pulseshape, with each pulse having asubstantially constant instantaneous irradiance over the pulse ON time.In certain embodiments, the peak irradiances of the pulses differ fromone another, while in certain other embodiments, the peak irradiances ofthe pulses are substantially equal to one another (see, e.g., FIGS. 21Aand B, and 21C and D, respectively, of United States Patent PublicationNo. 2009/0254154, each of which is incorporated in its entirety byreference herein). Various other temporal pulseshapes (e.g., triangular,trapezoidal) are also compatible with certain embodiments describedherein. For example, FIG. 21C of U.S. patent application Ser. No.12/403,824, filed Mar. 13, 2009, which is incorporated in its entiretyby reference herein, schematically illustrates a plurality oftrapezoidal pulses in which each pulse has a rise time (e.g.,corresponding to the time between an instantaneous irradiance of zeroand a peak irradiance of the pulse) and a fall time (e.g., correspondingto the time between the peak irradiance of the pulse and aninstantaneous irradiance of zero). In certain embodiments, the rise timeand the fall time can be expressed relative to a specified fraction ofthe peak irradiance of the pulse (e.g., time to rise/fall to 50% of thepeak irradiance of the pulse).

As used herein, the term “peak irradiance” of a pulse P_(i) has itsbroadest reasonable interpretation, including but not limited to, themaximum value of the instantaneous irradiance I(t) during the temporalpulsewidth of the pulse. In certain embodiments, the instantaneousirradiance is changing during the temporal pulsewidth of the pulse whilein certain other embodiments, the instantaneous irradiance issubstantially constant during the temporal pulsewidth of the pulse.

As used herein, the term “pulse irradiance” I_(P) _(i) of a pulse P_(i)has its broadest reasonable interpretation, including but not limitedto, the integral of the instantaneous irradiance I(t) of the pulse P_(i)over the temporal pulsewidth of the pulse:

I_(P_(i)) = ∫_(T_(i))^(T_(i + 1))I(t) ⋅ dt/(T_(i + 1) − T_(i)).As used herein, the term “total irradiance” I_(TOTAL) has its broadestreasonable interpretation, including but not limited to, the sum of thepulse irradiances of the pulses:

$I_{TOTAL} = {\sum\limits_{i = 0}^{N}{I_{P_{i}}.}}$As used herein, the term “time-averaged irradiance” I_(AVE) has itsbroadest reasonable interpretation, including but not limited to, theintegral of the instantaneous irradiance I(t) over a period of time Tlarge compared to the temporal pulsewidths of the pulses:

I_(AVE) = ∫₀^(T)I(t) ⋅ dt/T.The integral

∫₀^(T)I(t) ⋅ dtprovides the energy of the pulsed light beam.

For example, for a plurality of square pulses with different pulseirradiances I_(P) _(i) and different temporal pulsewidths ΔT_(i), thetime-averaged irradiance over a time T equals

$I_{AVE} = {\frac{1}{T}{\sum\limits_{i}{{I_{P_{i}} \cdot \Delta}\;{T_{i}.}}}}$For another example, for a plurality of square pulses with equal pulseirradiances I_(P), with equal temporal pulsewidths, and equal pulse OFFtimes (having a duty cycle D), the time-averaged irradiance equalsI_(AVE)=I_(P)·D. For example, as shown in FIG. 21D of U.S. patentapplication Ser. No. 12/403,824, the time-averaged irradiance (shown asa dashed line) is less than the pulse irradiance of the pulses.

The pulse irradiances and the duty cycle can be selected to provide apredetermined time-averaged irradiance. In certain embodiments in whichthe time-averaged irradiance is equal to the irradiance of acontinuous-wave (CW) light beam, the pulsed light beam and the CW lightbeam have the same number of photons or flux as one another. Forexample, a pulsed light beam with a pulse irradiance of 5 mW/cm² and aduty cycle of 20% provides the same number of photons as a CW light beamhaving an irradiance of 1 mW/cm². However, in contrast to a CW lightbeam, the parameters of the pulsed light beam can be selected to deliverthe photons in a manner which achieve results which are not obtainableusing CW light beams.

For example, for hair removal, tattoo removal, or wrinkle smoothing,pulsed light beams have previously been used to achieve selectivephotothermolysis in which a selected portion of the skin is exposed tosufficiently high temperatures to damage the hair follicles (e.g.,temperatures greater than 60 degrees Celsius), to ablate the tattoo ink(e.g., temperatures much greater than 60 degrees Celsius), or to shrinkthe collagen molecules (e.g., temperatures between 60-70 degreesCelsius), respectively, while keeping the other portions of skin atsufficiently low temperatures to avoid unwanted damage or discomfort.The parameters of these pulsed light beams are selected to achieve thedesired elevated temperature at the selected portion of the skin byabsorption of the light by the selected chromophore while allowing heatto dissipate (characterized by a thermal relaxation time) during thepulse OFF times to keep other areas of skin at lower temperatures. Asdescribed by J. Lepselter et al., “Biological and clinical aspects inlaser hair removal,” J. Dermatological Treatment, Vol. 15, pp. 72-83(2004), the pulse ON time for hair removal is selected to be between thethermal relaxation time for the epidermis (about 3-10 milliseconds) andthe thermal relaxation time for the hair follicle (about 40-100milliseconds). In this way, the hair follicle can be heated tosufficiently high temperatures to damage the follicle without causingexcessive damage to the surrounding skin.

In contrast to these treatments which are based on creating thermaldamage to at least a portion of the skin, certain embodiments describedherein utilize pulse parameters which do not create thermal damage to atleast a portion of the skin. In certain embodiments, one or more of thetemporal pulsewidth, temporal pulseshape, duty cycle, repetition rate,and pulse irradiance of the pulsed light beam are selected such that noportion of the skin is heated to a temperature greater than 60 degreesCelsius, greater than 55 degrees Celsius, greater than 50 degreesCelsius, or greater than 45 degrees Celsius. In certain embodiments, oneor more of the temporal pulsewidth, temporal pulseshape, duty cycle,repetition rate, and pulse irradiance of the pulsed light beam areselected such that no portion of the skin is heated to a temperaturegreater than 30 degrees Celsius above its baseline temperature, greaterthan 20 degrees Celsius above its baseline temperature, or greater than10 degrees Celsius above its baseline temperature. In certainembodiments, one or more of the temporal pulsewidth, temporalpulseshape, duty cycle, repetition rate, and pulse irradiance of thepulsed light beam are selected such that no portion of the bone marrowis heated to a temperature greater than 5 degrees Celsius above itsbaseline temperature, greater than 3 degrees Celsius above its baselinetemperature, or greater than 1 degree Celsius above its baselinetemperature. As used herein, the term “baseline temperature” has itsbroadest reasonable interpretation, including but not limited to, thetemperature at which the tissue would have if it were not irradiated bythe light. In contrast to previous low-light level therapies, the pulsedlight beam has an average radiant power in the range of about 1 Watt toabout 6 Watts including about 1 to about 3 Watts, about 3 to about 4Watts, or about 4 Watts to about 6 Watts.

In certain embodiments, the pulse parameters are selected to achieveother effects beyond those which are achievable using CW light beams. Incertain embodiments described herein, pulsed irradiation may provide amore efficacious mobilization of HSCs, increased proliferation,differentiation, engraftment of stem cells, or improve the overallefficacy of stem cell therapy. The pulsed irradiation can provide higherpeak irradiances for shorter times, thereby providing more power topropagate to the target tissue while allowing thermal relaxation of theintervening tissue and blood between pulses to avoid unduly heating theintervening tissue. The time scale for the thermal relaxation istypically in the range of a few milliseconds. For example, the thermalrelaxation time constant (e.g., the time for tissue to cool from anelevated temperature to one-half the elevated temperature) of human skinis about 3-10 milliseconds, while the thermal relaxation time constantof human hair follicles is about 40-100 milliseconds. Thus, previousapplications of pulsed light to the body for hair removal have optimizedtemporal pulsewidths of greater than 40 milliseconds with time betweenpulses of hundreds of milliseconds.

However, while pulsed light of this time scale advantageously reducesthe heating of intervening tissue and blood, it does not provide anoptimum amount of efficaciousness as compared to other time scales. Incertain embodiments described herein, one or more of the patient's longbones are irradiated with pulsed light having parameters which are notoptimized to reduce thermal effects, but instead are optimized tostimulate, to excite, to induce, or to otherwise support one or moreintercellular or intracellular biological processes which are involvedin the mobilization and proliferation of HSCs from the bone marrowcavity. Thus, in certain such embodiments, the selected temporal profilecan result in temperatures of the irradiated tissue which are higherthan those resulting from other temporal profiles, but which are moreefficacious than these other temporal profiles. In certain embodiments,the pulsing parameters are selected to utilize the kinetics of thebiological processes rather than optimizing the thermal relaxation ofthe tissue. In certain embodiments, the pulsed light beam has a temporalprofile (e.g., peak irradiance per pulse, a temporal pulse width, and apulse duty cycle) selected to modulate membrane potentials in order toenhance, restore, or promote cell survival, cell function, or both ofthe irradiated target cells. For example, in certain embodiments, thepulsed light has a temporal profile which enhances the proliferation ofHSCs but does not optimize the thermal relaxation of the irradiatedtissue.

In certain embodiments, the temporal profile (e.g., peak irradiance,temporal pulse width, and duty cycle) are selected to enhance theproliferation and mobilization of HSCs from the bone marrow whilemaintaining the irradiated portion of the long bones at or below apredetermined temperature. This predetermined temperature is higher thanthe optimized temperature which could be achieved for other temporalprofiles (e.g., other values of the peak irradiance, temporal pulsewidth, and duty cycle) which are optimized to minimize the temperatureincrease of surrounding tissue due to the irradiation. For example, atemporal profile having a peak irradiance of 10 W/cm² and a duty cycleof 20% has a time-averaged irradiance of 2 W/cm². Such a pulsed lightbeam provides the same number of photons to the irradiated surface asdoes a continuous-wave (CW) light beam with an irradiance of 2 W/cm².However, because of the “dark time” between pulses, the pulsed lightbeam can result in a lower temperature increase than does the CW lightbeam. To minimize the temperature increase of the irradiated portion ofthe long bones, the temporal pulse width and the duty cycle can beselected to allow a significant portion of the heat generated per pulseto dissipate before the next pulse reaches the irradiated portion. Incertain embodiments described herein, rather than optimizing the beamtemporal parameters to minimize the temperature increase, the temporalparameters are selected to effectively correspond to or to besufficiently close to the timing of the biomolecular processes involvedin the absorption of the photons to provide an increased efficacy.Rather than having a temporal pulse width on the order of hundreds ofmicroseconds, certain embodiments described herein utilize a temporalpulse width which does not optimize the thermal relaxation of theirradiated tissue (e.g., milliseconds, tens of milliseconds, hundreds ofmilliseconds). Since these pulse widths are significantly longer thanthe thermal relaxation time scale, the resulting temperature increasesare larger than those of smaller pulse widths, but still less than thatof CW light beams due to the heat dissipation the time between thepulses.

Beam Size and Beam Profile

In certain embodiments, the light beam will be manipulated (e.g. withnon-transmissive materials) to yield a rectangular, oval, or othergeometric shape in the approximate length and width of the particularlong bone to be irradiated. In certain embodiments, multiple lightsources can be used to irradiate a single long bone.

In certain embodiments, the light beam has a nominal diameter in a rangeof about 10 millimeters to about 40 millimeters, in a range of about 20millimeters to about 35 millimeters, less than 33 millimeters, or equalto about 30 millimeters. In certain embodiments, the cross-sectionalarea is generally circular with a radius in a range of about 1centimeter to about 2 centimeters. In certain embodiments, the lightbeam irradiating the skin has a cross-sectional area greater than about2 cm² or in a range of about 2 cm² to about 20 cm² (e.g., at an emissionsurface of an optical element generating the light beam).

As used herein, the beam diameter is defined to be the largest chord ofthe perimeter of the area of the skin irradiated by the light beam at anintensity of at least 1/e² of the maximum intensity of the light beam.The perimeter of the light beam used to determine the diameter of thebeam is defined in certain embodiments to be those points at which theintensity of the light beam is 1/e² of the maximum intensity of thelight beam. The maximum-useful diameter of certain embodiments islimited by the size of the patient's long bones and by the heating ofthe patient's body by the irradiation. The minimum-useful diameter ofcertain embodiments is limited by heating and by the total number oftreatment sites that could be practically implemented. For example, tocover a large area of one of the patient's long bones with a beam havinga small beam diameter would correspondingly use a large number oftreatment sites. In certain embodiments, the time of irradiation pertreatment site can be adjusted accordingly to achieve a desired exposuredose.

Specifying the total flux inside a circular aperture with a specifiedradius centered on the exit aperture (“encircled energy”) is a method ofspecifying the power (irradiance) distribution over the light beamemitted from the emission surface of a light source. The “encircledenergy” can be used to ensure that the light beam is not tooconcentrated, too large, or too small. In certain embodiments, the lightbeam emitted from the emission surface has a total radiant power, andthe light beam has a total flux inside a 20-millimeter diametercross-sectional circle centered on the light beam at the emissionsurface which is no more than 75% of the total radiant power. In certainsuch embodiments, the light beam has a total flux inside a 26-millimeterdiameter cross-sectional circle centered on the light beam at theemission surface 22 which is no less than 50% of the total radiantpower.

In certain embodiments, the beam intensity profile has a semi-Gaussianprofile, while in certain other embodiments, the beam intensity profilehas a “top hat” profile. In certain embodiments, the light beam issubstantially without high flux regions or “hot spots” in the beamintensity profile in which the local flux, averaged over a 3 millimeterby 3 millimeter area, is more than 10% larger than the average flux.Certain embodiments employ a light beam substantially without hot spots,thereby avoiding large temperature gradients at the patient's skin whichwould otherwise cause discomfort to the patient.

Divergence

In certain embodiments, the beam divergence emitted from the emissionsurface of the light source is significantly less than the scatteringangle of light inside the body tissue being irradiated, which istypically several degrees. In certain embodiments, the light beam has adivergence angle greater than zero and less than 35 degrees.

Targets of Cell Therapy

As discussed above, cell therapy can be used to treat a wide variety ofdisorders. For example, Parkinson's disease is a chronic, progressiveneurodegenerative disease or movement disorder that affects up to onemillion people in the United States. Parkinson's disease affectsneurologic function by degrading motor skills of the subject and bycausing dementia. The pathology of Parkinson's disease includes reducedformation and action of dopamine, which is produced in the dopaminergicneurons of the brain. Previous research of the causes and possibletreatments of Parkinson's disease have been directed towards efforts tocompensate for the reduced formation and action of dopamine caused bythe disease.

Dementia (e.g., as resulting from Parkinson's disease) is a collectionof symptoms but is not generally considered a disease itself. Dementiais characterized as the loss of cognitive function having a severity soas to interfere with a person's daily activities. Cognitive functionincludes activities such as knowing, thinking, learning, memory,perception, and judging. Symptoms of dementia can also include changesin personality, mood, and behavior of the subject. Although, in somecases, dementia can be treated by treating or curing the underlyingdisease (e.g. infection, nutritional deficiency, tumor), in most casesdementia is considered incurable.

Dementia tends to develop mostly in elderly people. It has beenestimated that 5-8% of all people over the age of 65 have some form ofdementia, and with that figure doubling every five years above that age.It is estimated that as many as half of people in their 80's suffer fromsome form of dementia. One of the most common causes of dementia is anunderlying neurological disorder, such as Alzheimer's disease.Alzheimer's disease affects about 4 million Americans and appears to beincreasing in frequency, as is the resulting dementia. Othernon-limiting examples of causes of dementia include AIDS or HIVinfection, Creutzfeldt-Jakob disease, head trauma (includingsingle-event trauma and long term trauma such as multiple concussions orother traumas which may result from athletic injury), Lewy body disease,Pick's disease, Parkinson's disease, Huntington's disease, drug oralcohol abuse, brain tumors, hydrocephalus, and kidney or liver disease.

Furthermore, people suffering from mental diseases or disorders cansuffer from varying levels of diminishment of cognitive function that donot rise to the level of dementia. Additionally, generally healthyindividuals may also perceive some loss of cognitive function, mostcommonly a reduction in the function of memory. Loss or diminishment ofmemory may occur in any of the four commonly designated phases ofmemory, namely learning, retention, recall and recognition, and may berelated to immediate memory, recent memory or remote memory. Loss ofmotor function may occur as a result of any of a number of causes,including many of those discussed above for which there is also a lossof cognitive function.

Alzheimer's disease is another neurological disorder that affectsnumerous individuals around the world. Replacement of diseased neuronsthrough cell therapy may help slow or compensate for the loss offunction Alzheimer's patients' experience.

Apart from degenerative disorders, acute injury to neural tissue maylead to loss of neural function. For example, traumatic brain injury canyield cell damage or death by both primary and secondary mechanisms(discussed further below). Head injury in general, whether from a directimpact to the head or from swelling of the brain due to indirect impact,can also reduce the function of neurons. Additionally, spinal cordinjury is one of the most publicly recognized forms of acute injury toneural tissue. Cell therapy in this area of neurological disorder isaimed at restoration (complete or partial) function to organs or limbsthat have lost function due to an injury. Even modest clinicalimprovements have the potential to yield great effects in terms ofrestoration of the activities an individual can perform as well as theirquality of life.

In addition to neurological disorders and injury, in severalembodiments, cell therapy plays a major role in treating cardiac damage.Myocardial infarctions and strokes often result in substantial loss offunction in portions of the myocardium. Generally, the myocardium isviewed as comprising a terminally differentiated group of cells withlimited capacity for self-renewal. Thus, cell therapy to treat cardiactissue damage represents great potential progress in helpingpost-infarction or post-stroke patients regain functionality.

Many other diseases that affect various tissues are key targets for celltherapy. In several embodiments, liver damage or cancer is treated withcell therapy in order to replace lost or malfunctioning cells. Inseveral embodiments, diabetic patients are treated with pancreaticprogenitor cells (or other stem cells differentiated to pancreaticidentity) in order to recapitulate loss of insulin secretion.

As discussed above, cancers are a particularly interesting area from theperspective of cell therapies, in that the standard therapeutic regimefor treating the disease, induces damage to host tissues. In the end,the goal is to kill the cancerous cells, and not so many of the hostcells that the patient dies. As such, combating these treatment-inducedside effects with cell therapy may increase the probability of survivalof cancer patients.

As discussed more fully below, the use of LLLT in combination with celltherapy enhances the efficacy of the cell therapy and leads to a morepronounced therapeutic effect. Moreover, LLLT, in several embodiments,positively impacts stem cells in a fashion which makes them moresuitable for use in cell therapy. Additionally, LLLT, in severalembodiments, not only enhances the effects of exogenously administeredstem cells used in cell therapy, it also positively affects endogenousstem cells (e.g., resident neural progenitors or resident cardiacprogenitors) such that the combination of administered cell andendogenous cells yields a synergistically enhanced therapeutic effect.

Embryonic stem cells, which are typically derived from an early stageembryo, have the potential to develop into any type of cell in the body.In contrast, adult stem cells generally develop into cell types relatedto the tissue from which the stem cells were isolated.

Use of embryonic stem cells in a clinical setting is often problematicbecause embryonic stem cells are typically allogeneic to a patient, asthe embryonic stem cells rarely originate from that patient. As aresult, rejection of transplanted embryonic stem cells may be asignificant concern. Likewise, the pluripotency of embryonic stem cellsdoes not guarantee differentiation into cells related to the targettissue. As discussed further below, in some embodiments, mesenchymalstem cells are used in order to limit the adverse immunological responseto transplanted cells. In contrast, adult stem cells taken from thepatient and subsequently reintroduced into the same patient willgenerally not be rejected. Further, because adult stem cells generallydevelop into related cell types, the risk that the adult stem cells willdevelop into undesired cell types may be reduced by taking adult stemcells from the tissue that is to be treated or repaired. Therefore, insome embodiments, non-embryonic stem cells are used (e.g., adult stemcells). In certain embodiments, use of autologous adult stem cells ispreferred in order to significantly minimize the risk of immunerejection of transplanted cells. However, in several embodiments,embryonic stem cells are employed.

LLLT and Stem Cell Mobilizing Compounds to Increase Stem Cell Production

In several embodiments, a method for increasing the number of one ormore particular cell types in a patient, such as in a patient'sbloodstream is disclosed. The cell types can include, for example, whiteblood cells (e.g., a neutrophil, macrophage, natural killer cell,basophil, eosinophil, B cell, CD4 T-cell, or CD8 T-cell) platelets, orred blood cells. In some embodiments, an increased quantity of cells,such as stem cells (e.g., HSC) are mobilized into the peripheralbloodstream of a patient for collection for a diagnostic purpose, suchas screening for a hematologic or oncologic disease, a therapeuticpurpose, such as autologous or heterologous blood or blood componentdonation, or to stimulate the bone marrow prior to transplantation, or astorage purpose, such as cell banking.

In some embodiments, a combination of a therapeutically-effective amountof one, two, or more agents that can stimulate mobilization into theperipheral bloodstream, production and/or improve function of one, two,or more cell types is administered. The agent(s) could be given throughany desired route of administration, including orally, rectally,intravenously, intramuscularly, subcutaneously, or an aerosol. Somenon-limiting embodiments of an agent that can stimulate mobilizationinto the peripheral bloodstream, production of and/or improve functionof a cell type include IL-1, IL-2, IL-3, IL-6, GM-CSF, G-CSF,plerixafor, PDGF, TGF-beta, NGF, IGFs, growth hormone, erythropoietin,thrombopoietin, and the like. In addition to naturally occurring growthfactors, growth factor analogs and growth factor derivatives such asfusion proteins can be used as well. In some embodiments, the methodinvolves administration of a therapeutically-effective amount of G-CSFand a therapeutically-effective amount of electromagnetic radiation. Insome embodiments, the method comprises administering a combination of atherapeutically-effective amount of plerixafor and atherapeutically-effective amount of electromagnetic radiation. In someembodiments, a therapeutically-effective amount of electromagneticradiation is combined with another agent that, in some embodiments,could be a hematopoietic stem cell mobilizer. In some embodiments, atherapeutically-effective amount of electromagnetic radiation iscombined with combinations of two or more of G-CSF, GM-CSF, plerixafor,IL-1, IL-2, IL-3, IL-6, PDGF, TGF-beta, NGF, IGFs, growth hormone,erythropoietin, thrombopoietin or another agent.

In some embodiments, the G-CSF and electromagnetic radiation are used toprevent or treat neutropenia. In still other embodiments, thetherapeutic agent and electromagnetic radiation are used to prevent ortreat anemia or thrombocytopenia.

In some embodiments, at least a portion of the electromagnetic radiationis administered after the administration of the agent, such as G-CSF. Inother embodiments, at least a portion of the electromagnetic radiationis administered prior to the agent, such as G-CSF. In still otherembodiments, at least a portion of the agent, such as G-CSF and at leasta portion of the electromagnetic radiation are administeredconcurrently. In some embodiments, the dose of G-CSF administered can bebetween about 1-200 micrograms/kg, such as between about 1-200micrograms/kg, 1-10 micrograms/kg, or 5-10 micrograms/kg intravenously(IV) or subcutaneously (SQ) every other day, every day, twice daily, oranother dosing frequency depending on the desired clinical result. Insome embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses of eitherthe G-CSF, electromagnetic radiation, or both are administered to thepatient until the patient's absolute neutrophil count increases togreater than 500/mm³, 750/mm³, 1000/mm³, 1250/mm³, 1500/mm³, or more.

In some embodiments, at least a portion of the electromagnetic radiationis delivered to one, two, or more bones including the long bones of thebody. The long bones of the body are those that are typically longerthan they are wide. Such bones include, but are not limited to thefemur, tibia, fibula, humerus, radius, ulna, metacarpals, andmetatarsals. Other non-limiting examples of bones to deliver theelectromagnetic radiation to could include the anterior or posterioriliac crest, ischial tuberosity, ribs, sternum, or cervical, thoracic,lumbar, or sacral vertebrae.

In several embodiments, the time-averaged irradiance at the HSCsreceiving light (e.g., the marrow cavity of the irradiated long bones)is greater than 0.01 mW/cm².

In several embodiments, the biostimulatory effect of phototherapy willenhance the effects of a particular agent such as G-CSF, plerixafor, oranother HSC mobilizer, on proliferation of a cell such as a HSC and/orproduction of neutrophils. Further examples of the biostimulatory effectof phototherapy are disclosed in U.S. Patent Application Publication No.2008/0221211, now abandoned, which describes the treatment ofneurological injury or cancer by administration of dicholoroacetic acidand/or electromagnetic radiation, and which is incorporated in itsentirety by reference herein. The efficacy of such a combinationapproach would be of great value to the medical field because HSCs couldbe collected peripherally. Based on the current skill and knowledge inthe art, the synergistic effect of the combination of an agent such asthose disclosed herein, e.g., a hematopoietic stem cell mobilizer andbiostimulation via phototherapy would also be unexpected, and wouldotherwise be thought to be contraindicated in some cancer or neutropenicpatients (the target patient population), as lighttherapy/electromagnetic radiation has been shown to induce proliferationof cancer cells.

LLLT Effects on Stem Cells

The LLLT devices, parameters, and procedures disclosed herein, inseveral embodiments, are used to enhance the efficacy of cell therapy.As discussed above, cell therapy is a growing field of medicine and incombination with the enhancing effects of LLLT, is an important additionto the selection of therapies to combat disease and injury that causecell death and/or loss of function. The term “progenitor cell” as usedherein has its broadest reasonable meaning, including but not limited to(1) a pluripotent, or lineage-uncommitted, progenitor cell, a “stemcell” or “mesenchymal stem cell” (MSC), that is potentially capable ofan unlimited number of mitotic divisions to either renew its line or toproduce progeny cells that will differentiate into any of a variety ofcells (e.g., cells of the central nervous system including neural cellssuch as astrocytes, oligodendrocytes, and neurons; cardiac cells;hematopoietic cells, etc.); or (2) a lineage-committed progenitor cellproduced from the mitotic division of a stem cell which will eventuallydifferentiate into a neural cell (or other cell type within the lineageof the stem cell, e.g., a cardiac progenitor cell differentiating into acardiomyocyte). Unlike the stem cell from which it is derived, thelineage-committed progenitor is generally considered to be incapable ofan unlimited number of mitotic divisions and will eventuallydifferentiate into a cell type within its lineage (e.g., neural toneural, cardiac to cardiac, etc.). Progenitor cells also encompass theendogenous stores of cells within the body that, in some embodiments arepositively affected by LLLT. For example, the resident bone marrow cellsare, in some embodiments, induced to proliferate and/or differentiate inresponse to LLLT administration. As described more fully above, inseveral embodiments, this response is advantageously used to boost theproduction of hematopoietic stem cells from the bone marrow to combatvarious leukemias or as a prophylactic measure prior to other cancertherapies.

The selection of progenitor cell type is, in several embodiments, drivenby the characteristics of the disease to be treated and the patient tobe treated. For example, certain cell types, such as bone marrow derivedstem cells are fairly easily isolated from a patient and readministeredto the same patient (e.g., autologous transplant to a cancer patientpost-therapy). As discussed above, LLLT is used, in some embodiments, toincrease the mobilization of bone marrow derived stem cells, therebyincreasing the efficiency of peripheral blood collection of stem cells.While in some embodiments, bone marrow derived stem cells areadministered in an allogeneic transplant, certain cell types may presentadditional benefits for such transplants. For example, mesenchymalcells, in several embodiments, are preferred due to their immunemodulating function, which is advantageous in an allogeneic transplantsetting.

Mesenchymal stem cells are typically isolated from bone marrow oradipose tissue, and, in several embodiments, suppress immune responses.In several embodiments, MSCs inhibit host T cell responses to a“non-self” marker (such as an allogeneic cell). In several embodiments,host antigen presenting cell (APC) function is altered by MSCs. In someembodiments, monocyte function is reduced by MSCs, through MSC-derivedinhibition of on monocyte maturation. In several embodiments, theseeffects of MSCs are manifest as decreased inflammation post-transplant.In some embodiments, the administration of MSCs assists in loweringinflammation due to disease or injury, in addition to reducingsubsequent inflammation due to the transplant of the cells themselves.In several embodiments, MSCs additionally function in an anti-fibroticmanner, which reduces fibrosis (and associated loss of function) in thetarget tissue. In some embodiments, the release of trophic/paracrinefactors (as discussed below) mediates such functions. Thus, theimmunomodulatory effects of MSCs are particularly advantageous inembodiments involving allogeneic cell therapy, as the MSCs assist incombating and/or reducing graft versus host immune responses which lowerthe efficacy of cell therapy.

As used herein, the term “viability” shall be given its ordinary meaningand shall also refer to the ability of a cell, be it a stem cell or aresident cell, to survive disease, trauma, or other injury that wouldcompromise the normal functionality of the cell. In some embodiments,viability is measured by assessing the size of a certain population ofcells, while in some embodiments, specific chemical, biological, oranalytical tests are performed to evaluate the viability of the cells.Viability is also, in some embodiments, assessed by function, wherein anincrease in function may be associated with an increase in viability.

As used herein, the term “proliferation” shall be given its ordinarymeaning and shall refer to the process by which one or more stem cells(endogenous or exogenous) divide and increase the population of stemcells (e.g., mitotic division). In several embodiments, proliferation ismeasured by simple total cell count. In other embodiments, proliferationis assessed by expression of certain proteins (e.g., proliferating cellnuclear antigen, PCNA), or by monitoring entry of cells into the cellcycle.

The term “differentiation” as used herein has its broadest reasonablemeaning, including but not limited to the process whereby anunspecialized, pluripotent stem cell proceeds through one or moreintermediate stage cellular divisions, ultimately producing one or morespecialized cell types. Differentiation thus includes the processwhereby precursor cells, e.g., uncommitted cell types that precede thefully differentiated forms but may or may not be true stem cells,proceed through intermediate stage cell divisions to ultimately producespecialized cell types. Differentiation encompasses the process wherebymesenchymal stem cells (MSC) are induced to differentiate into one ormore of the committed cell types comprising the central nervous system,in vivo or in vitro.

The term “migration” as used herein shall be given its ordinary meaningand shall also refer to the movement of a stem cell (either endogenousor exogenous) from its initial site (e.g., an endogenous storage site ora site of administration) to a second site (e.g., a final position in atarget tissue). In some embodiments, migration occurs based on fluidflow or pressure changes in the environment surrounding the cell. Insome embodiments, chemoattractant or chemorepellents induce migration ofcells.

As used herein, the term “engraftment” shall be given its ordinarymeaning and shall also refer to the process (or result of that process)whereby a cell is incorporated into another group of cells or anothertissue. For example, in some embodiments, exogenously administered stemcells engraft (e.g., become a part of) the host myocardium. Engraftmentmay or may not occur in conjunction with migration, depending on theembodiment. Likewise, engraftment may or may not be associated withincreased viability (e.g., engraftment is not a requirement formaintaining or increasing viability of cells), depending on theembodiment.

The terms “growth chamber” and “cell culture chamber” as used herein areused interchangeably and are to be interpreted very broadly to refer toany container or vessel suitable for culturing cells, including, but notlimited to, dishes, culture plates (single or multiple well),bioreactors, incubators, and the like. Certain embodiments describedherein utilize a cell culture apparatus such as is described in U.S.patent application Ser. No. 10/700,355, filed Nov. 3, 2003 andincorporated by reference herein in its entirety.

Implantation of Irradiated Cells

In several embodiments, LLLT is administered in combination with celltherapy, resulting in enhanced effects of cell therapy. The stem cellsmay be delivered by numerous routes, including direct injection,catheter-based approaches, intravascular administration,stereotactic-guided delivery, etc. In some embodiments, the enhancementis manifest as an increased viability of the stem cells. In someembodiments, the increased viability is advantageous because the cellsare present in a tissue that is afflicted with a disease, and thereforemay present one or more cellular pro-death signals. In some embodiments,LLLT enhances viability by increasing the resistance of the cells toapoptotic factors. In some embodiments, anti-apoptotic pathways areupregulated in cells that have received LLLT. While not the only factorto consider, enhanced viability of cells, in some embodiments, is ofparticular importance, as replacement of damaged or diseased cells maybe the most efficacious when the replacement cell is likely to survive.

In some embodiments, LLLT enhances the proliferation of stem cells. Insome embodiments, the increase in proliferation results in asubstantially larger population of stem cells that can functionallyreplace (partially or fully) the damaged or diseased cells of the host.For example, administration of a small population of neural progenitorcells to a individual with Parkinson's disease (e.g. by stereotacticdelivery of the cells to a target region of the brain) andadministration of LLLT, in some embodiments, induces proliferation ofthe neural progenitor cells to a degree which compensates for the lossof speech or motor control associated with Parkinson's. In someembodiments, LLLT can enhance the proliferation of endogenous stem cellsto potentiate the effects of the exogenously delivered stem cells. Insome embodiments, the light parameters discussed above are tailored togenerate a desired proliferative growth curve (e.g., decreasingfrequency and/or intensity of LLLT over time to reduce the proliferativestimulation). In this manner, uncontrolled proliferation of eitherendogenous or administered stem cells (or other cells receiving LLLT) isavoided.

In several embodiments, LLLT improves the migration of stem cells. Insome embodiments, chemoattractant signals present in the target tissueinduce the migration of stem cells (endogenous or exogenous) to adesired location. In several embodiments, LLLT potentiates the responseof the cells to such a signal, thereby allowing a more rapidrepositioning of cells to a desired location. Once in its desiredlocation, in some embodiments, the additional effects of LLLT describedherein allow the cell to more rapidly or efficiently provide atherapeutic benefit (e.g., provide function that is lost due to damageor disease). In some embodiments, chemorepellant signals drive cellsaway from an undesired location. In some embodiments, the combination ofchemoattractant and chemorepellant signals work in concert to direct thecells to a desired location. In some embodiments, the chemoattractantand/or chemorepellant signals are exogenously administered as well. Insome such embodiments, a series of injections of a chemoattractantcompound are pre-delivered to a target tissue in order to generate agradient of signal for the administered cells to respond to.Post-administration, the cells migrate along this gradient, therebycoming to rest at a desirable position.

In several embodiments, engraftment of the stem cells is improved byadministration of LLLT. As discussed above this may be particularlyadvantageous in certain therapeutic applications, such as for examplecell therapy directed to target tissues that experience shear flow,flex, or other forces that may dislodge the cells. For example, in someembodiments, LLLT and cardiac progenitor cells are administered, andengraftment is enhanced (as compared to progenitor cells alone). This isparticularly advantageous because the blood flow through the heart couldwash the administer cells out of the target organ (for example if thetarget for the cells was an intracardiac site). Moreover, the constantflex of the myocardium may dislodge the administered cells. As such, theincreased engraftment of administered cells increases the efficacy ofthe therapy due to the retention of a larger number of cells at thetarget site.

In several embodiments, the function of stem cells is improved by theadministration of LLLT. Functional assessment depends on the variety ofcell that is administered, e.g., neural function versus cardiacfunction. Modes of assessing are described in more detail below. Inseveral embodiments, however, the function of administered stem cells(or endogenous stem cells) is improved by light therapy. By way ofexample, LLLT may promote increase firing of a neuron (derived from aneural progenitor). Similarly, in some embodiments, increasedneurotransmitter release results. In some embodiments, alterations incell biology occur (e.g., increased or decreased axonal transport) whichare beneficial to the function of the neuron.

In several embodiments, LLLT positively impacts the administered cellswhich are themselves enhanced in one or more of the manners describedherein. In some embodiments, the effects of the LLLT on the stem cellsresults in a cascade that yields beneficial effects to the cells of thedamaged or diseased host tissue. For example, in some embodiments, theirradiation of stem cells with LLLT induces pro-survival paracrinefactor (e.g., growth factors, immunosuppressive molecules) release fromthe stem cells, which, in turn, improves the survival of the damaged ordiseased host tissue. Thus, in some embodiments, the characteristics ofthe stem cells are enhanced, which improves cell therapy. In someembodiments, the LLLT-treated stem cells become a source of a signalthat improves damaged or diseased host tissue (e.g., the cells are avehicle for a beneficial effect rather than providing the effectdirectly).

In several embodiments, the stem cells are responsive to the in vivoenvironment into which they are transplanted. For example, tissue damageor disease is often associated with various signaling cascades, which,in balance, determine the outcome of a subset of cells (or the entiretissue). In some embodiments, the administered cells detect, andsubsequently respond to the milieu of damage, disease, and/orinflammatory signals in the target tissue. In several embodiments (asdiscussed above), certain characteristics of the administered cellsadvantageously alter the balance, to the benefit of the survival of theadministered cells and/or the cells of the host tissue. For example, theMSCs discussed above, in several embodiments, respond to thepro-inflammatory environment in a damaged tissue by releasinganti-inflammatory cytokines, altering T-cell function, and/or alteringmonocyte maturation. Thus, MSCs may be of particular benefit inallogeneic transplants. Also, in some embodiments, other progenitor celltypes possess similar environmentally-responsive characteristics. Suchcells, with the ability to respond to local signals, generatecounteractive local and/or paracrine signals, and effectively alter thelocal environment in a beneficial (e.g., pro-survival or regeneration offunction manner) are used in several embodiments. In some embodiments, acombination of these mechanisms results. As discussed, such cells areparticularly advantageous in allogeneic transplants, though in someembodiments, they are used in autologous cell transplants.

In certain embodiments, a method is provided for treating damage orillness in the central nervous system in a mammal or human, comprisingdelivering an effective amount of light energy to an in vitro culturecomprising progenitor cells, (e.g. stem cells, induced pluripotentcells, genetically modified adult cells, etc.) and implanting the cellsinto the central nervous system of a mammal or human, wherein deliveringan effective amount of light energy includes delivering light having awavelength in the visible to near-infrared wavelength range and a powerdensity of at least about 0.01 mW/cm² to the cells in culture.

In certain embodiments, treatment of a patient comprises implantation ofprogenitor cells into the central nervous system (“CNS”) of the patient.Following implantation, the progenitor cells differentiate to form oneor more cell types of the central nervous system. The implanted cellsmay serve any of a variety of purposes, including replacement of cellsor tissues that have been irreparably damaged, repair of a portion ofthe CNS, enhance the production of important CNS neurochemicals such asdopamine, seratonin, endogenous opioid peptides, and the like.Implantation of progenitor cells may be performed alone, or it may bedone in combination with the methods of enhancing neurologicfunctioning, as described herein. For example, the progenitor cells maybe treated with an agent or combination of agents in addition to thelaser irradiation, prior to or after implantation. By way of example,the additional agent may be selected from the group consisting ofpharmaceutical compounds, cytokines, growth factors, neurotransmitters,hormones, trophic factors, transcription factors, monoclonal antibodies,polyclonal antibodies, venom, or signal transduction molecules. Inseveral embodiments, the agent or combination of agents may have theeffect of stimulating or mobilizing progenitor cells.

In several embodiments, treatment of a patient suffering from loss ofcardiac function due to an adverse cardiac event comprises implantationof cardiac progenitor cells into the myocardium of the patient (e.g., bycatheter-based injection into the myocardial wall). As discussed,herein, the cells may optionally be pre-irradiated, or may be irradiatedpost-implantation, or combinations thereof. Following implantation, theprogenitor cells differentiate to form one or more cardiac cell types.In some embodiments, the cells functionally replace the damaged cardiaccells of the patient, thereby restoring cardiac function (partially orfully). Adverse cardiac events include, but are not limited to,myocardial infarction, ischemic cardiac tissue damage, congestive heartfailure, aneurysm, atherosclerosis-induced events, cerebrovascularaccident (stroke), and coronary artery disease

In certain embodiments, progenitor cells are inoculated and grown in acell culture in vitro, using parameters including power density asdiscussed above. Because the light energy is applied directly to thecell culture in vitro and does not travel through intervening bodytissue, the power density selected to be delivered to the cell isgenerally equal to the power density of the light energy as it isemitted from the light apparatus. If lenses, filters, dispersiongratings, or any other material lies between the light source and thecells, any absorption or dispersion of the light energy by such materialshould be taken into account and the applied light energy adjusted, ifneeded, to account for the material. In certain embodiments, the treatedcells are implanted following treatment. In certain other embodiments,at least some treated cells remain in culture to maintain the cell linefor later use.

After in vitro treatment of cells using electromagnetic energy, thecells are transplanted or implanted to a recipient site in a patient. Incertain embodiments, the treatment prior to transplantation orimplantation includes culturing cells sufficient for implantation. Therecipient site may be a site of injury, illness, or defect, or it may bea region of relatively healthy tissue. In certain embodiments, therecipient site and/or the region surrounding such site is treated withlight energy according to the methods described supra, before and/orafter implantation to enhance the rate at which the implanted cells areintegrated with surrounding tissue at the recipient site.

In certain embodiments, progenitor cells such as stem cells are treatedwith electromagnetic energy as noted above and then implanted into thebrain of a patient, such a patient who is at risk for Parkinson'sdisease, exhibits symptoms of Parkinson's disease, and/or has beendiagnosed with Parkinson's disease. As discussed herein, numerous otherdiseases are treated with combination of appropriate stem cells andLLLT. Following implantation, the recipient site is optionally treatedwith electromagnetic energy, including directly at the recipient site orthrough the skull at the recipient site, or some other portion of thebrain or other neural tissue, such as the cortex or the spinal cord. Inseveral embodiments, the transplanted cells produce dopamine to treat,or lessen the symptoms and/or delay onset of Parkinson's disease in thepatient.

In certain embodiments, progenitor cells are treated withelectromagnetic energy and implanted or transplanted at a site ofphysical trauma to the spinal cord or one or more nerves of a patient.Following implantation, the recipient site is optionally treated withelectromagnetic energy. Such optional treatment may include treatmentimmediately following implantation and/or one or more treatment periodsfollowing implantation. The transplanted cells help repair damage to thespinal cord or nerve(s) such that the recovery or prognosis is enhancedin patients having implanted progenitor cells as compared with those whodo not receive such implants.

In several embodiments, the progenitor cells are treated withelectromagnetic energy after delivery to the target tissue (e.g., invivo). In some embodiments, this approach improves the overall efficacyof treatment, as there is limited lag time between the exposure of thecells to LLLT and the receipt of beneficial effects by the targettissue. In several embodiments, combinations of LLLT treatment are used.For example, in some embodiments, cells are irradiated both before andafter administration. In some embodiments, irradiation occurs one ormore times in vitro prior to implantation and/or one or more times afterimplantation.

Procedures for Light Therapy

In certain embodiments, a patient is treated by identifying a pluralityof treatment sites (e.g., at least about 10) on the patient's scalp orother target tissue, administering a plurality of stem cells, directingan electromagnetic radiation source to each of the treatment sites, andpropagating electromagnetic radiation from the source to each treatmentsite. In some embodiments, the stem cells are irradiated prior toadministration to a patient. In some embodiments, irradiation of thecells is performed prior to implantation as well as one or more timespost-implantation. In certain embodiments, the electromagnetic radiationfrom the source has a wavelength within a range between about 600 and1000 nanometers, including about 600 to 630 nm, 630 to 650, nm, 650 to670 nm, 670 to 700 nm, 700 to 800 nm, 800 to 900 nm, 900 to 1000 nm, andoverlapping ranges thereof.

In some embodiments a single treatment site is irradiated. In someembodiments, the treatment site is selected from the group consistingof, heart, lungs, liver, pancreas, kidney, spleen, intestine, bone, bonemarrow, teeth/gums, skeletal or smooth muscle, skin, or combinationsthereof.

As described more fully below, in certain embodiments, the treatmentsites are identified using an apparatus comprising a plurality ofoptically transmissive elements, each of which corresponds to atreatment site. In certain such embodiments, each of the treatment sitesis irradiated by electromagnetic radiation from a source placed incontact with each of the optically transmissive elements. In certainother embodiments, the treatment sites are instead identified by otherindicia. For example, each of the treatment sites can be identified bymarkings made on the scalp, or by structures placed in proximity to thescalp. Each of the treatment sites can then be irradiated. In certainembodiments, each of the treatment sites is irradiated by anelectromagnetic radiation source in contact with the scalp or in contactwith an intervening optically transmissive element which contacts thescalp. In certain other embodiments, the scalp is not contacted byeither the electromagnetic radiation source or an intervening element.

In certain embodiments, each of the treatment sites is irradiated usinga single electromagnetic radiation source which is sequentially movedfrom one treatment site to another. In certain other embodiments, aplurality of sources are used to irradiate multiple treatment sitesconcurrently. In certain such embodiments, the number of sources isfewer than the number of treatments sites, and the plurality of sourcesare sequentially moved to sequentially irradiate the treatment sites.

FIG. 33 is a flow diagram of an example method 700 for controllablyexposing at least one predetermined area of a patient's scalp to laserlight to irradiate the patient's brain. As discussed above, though notshown in FIG. 33, similar methods are used to treatment of othertissues, in some embodiments. Additionally not shown in FIG. 33 are theoptional time points for administering stem cells to a target tissue. Asdiscussed above, cell administration occurs prior to irradiation of thetissue (e.g. before 740). In other embodiments, administration of cellsis performed after irradiation of the tissue 740. In still otherembodiments, irradiation and/or cell delivery occurs multiple times overa therapeutic regime. As described more fully below, the method 700 isdescribed by referring to the wearable apparatus 500 and the lightemitting apparatus 600 described herein. Other configurations of awearable apparatus 500 and a light emitting apparatus 600 are alsocompatible with the method 700 in accordance with embodiments describedherein.

The method 700 comprises providing a light emitting apparatus 600 in anoperational block 710. In certain embodiments, the light emittingapparatus 600 comprises a source 610 of laser light, an optical conduit620 optically coupled to the source 610, and an optical device 630optically coupled to the optical conduit 620. Other configurations ofthe light emitting apparatus 600 besides those in FIGS. 28-34 are alsocompatible with certain embodiments described herein.

The method 700 further comprises placing a wearable apparatus 500 overthe patient's scalp (or other treatment site) in an operational block720. The apparatus 500 comprises a body 510 and a plurality of elements520. Each element 520 has a first portion 522 which conforms to acorresponding portion of the patient's scalp when the apparatus 500 isworn by the patient. Each element 520 also has a second portion 524which conforms to the optical device 630 when the optical device 630contacts the element 520. Each element 520 is substantially transmissiveto laser light emitted by the optical device 630. Other configurationsof the wearable apparatuses depicted in the Figures are also compatiblewith certain embodiments described herein.

The method 700 further comprises placing the light emitting apparatus600 in contact with an element 520 corresponding to at least a portionof the predetermined area of the patient's scalp (or other treatmentsite) to be irradiated in an operational block 730. The method 700further comprises irradiating the portion of the predetermined area ofthe patient's scalp with light emitted by the light emitting apparatus600 and transmitted through the element 520 in an operational block 740.As discussed above, depending on the embodiment, the stem cells areoptionally delivered prior to irradiating the target tissue. In someembodiments, the stem cells are irradiated after administration to thetarget tissue.

In certain embodiments, providing the light emitting apparatus 600 inthe operational block 710 comprises preparing the light emittingapparatus 600 for use to treat the patient. In certain embodiments,preparing the light emitting apparatus 600 comprises cleaning theportion of the light emitting apparatus 600 through which laser light isoutputted. In certain embodiments, preparing the light emittingapparatus 600 comprises verifying a power calibration of laser lightoutputted from the light emitting apparatus 600. Such verification cancomprise measuring the light intensity output from the light emittingapparatus 600 and comparing the measured intensity to an expectedintensity level.

In certain embodiments, placing the wearable apparatus 500 over thepatient's scalp in the operational block 720 comprises preparing thepatient's scalp (or other tissue) for treatment. For example, in certainembodiments, preparing the patient's scalp for treatment comprisesremoving hair from the predetermined areas of the patient's scalp to beirradiated. Removing the hair (e.g., by shaving) advantageously reducesheating of the patient's scalp by hair which absorbs laser light fromthe light emitting apparatus 600. In certain embodiments, placing thewearable apparatus 500 over the patient's scalp in the operational block720 comprises positioning the wearable apparatus 500 so that eachelement 520 is in contact with a corresponding portion of the patient'sscalp.

In certain embodiments, placing the light emitting apparatus 600 incontact with the element 520 in the operational block 730 comprisespressing the light emitting apparatus 600 to the element 520 so that thefirst portion 522 of the element 520 conforms to the patient's scalp andthe second portion 524 of the element 520 conforms to the light emittingapparatus 600. In certain embodiments, by pressing the light emittingapparatus 600 against the element 520 in this way, pressure is appliedto the portion of the patient's scalp in contact with the element 520 soas to advantageously blanch the portion of the patient's scalp incontact with the element 520.

In certain embodiments, irradiating the portion of the predeterminedarea of the patient's scalp (or other tissue) in the operational block740 comprises triggering the outputting of light from the light emittingapparatus 600 by pressing the light emitting apparatus 600 against theelement 520 with a predetermined level of pressure. In certainembodiments, the outputting of light from the light emitting apparatus600 continues only if a predetermined level of pressure is maintained bypressing the light emitting apparatus 600 against the element 520. Incertain embodiments, light is outputted from the light emittingapparatus 600 through the element 520 for a predetermined period oftime.

In certain embodiments, the method further comprises irradiatingadditional portions of the predetermined area of the patient's scalp (orother tissue) during a treatment process. For example, after irradiatinga first portion of the predetermined area corresponding to a firstelement 520, as described above, the light emitting apparatus 600 can beplaced in contact with a second element 520 corresponding to a secondportion of the predetermined area and irradiating the second portion ofthe predetermined area with light emitted by the light emittingapparatus 600 and transmitted through the element 520. The variousportions of the predetermined area of the patient's scalp can beirradiated sequentially to one another in a predetermined sequence. Incertain embodiments, the predetermined sequence is represented byindicia corresponding to the elements 520 of the wearable apparatus 500.In certain such embodiments, the laser emitting apparatus 600 comprisesan interlock system which interfaces with the indicia of the wearableapparatus 500 to prevent the various portions of the predetermined areafrom being irradiated out of the predetermined sequence.

In certain embodiments, a system for treating a patient comprises asupport (e.g., a wearable apparatus 500 as described herein) foridentifying a plurality of sites on a patient's scalp (or othertreatment site) for the application of therapeutic electromagneticenergy in a wavelength range between about 800 nanometers and about 830nanometers. The system further comprises an instruction for use of thesupport in combination with an electromagnetic light source (e.g., alight emitting apparatus 600 as described herein) of the therapeuticelectromagnetic energy. The instruction for use in certain embodimentscomprises instructions compatible with the method 700 described herein.

In certain embodiments, a system for treating a patient comprises anelectromagnetic light source (e.g., a light emitting apparatus 600 asdescribed herein) and a plurality of stem cells for administration to atarget tissue in the patient. The system further comprises aninstruction for use of the electromagnetic radiation source by opticallycoupling the source to a patient's scalp (or other site) at a pluralityof locations to deliver a therapeutic electromagnetic energy to thepatient's brain (or other tissue). The instruction for use in certainembodiments comprises instructions compatible with the method 700described herein.

Certain embodiments utilizing phototherapy as described herein are basedat least in part on the finding described above that, for a selectedwavelength, the power density (light intensity or power per unit area,in W/cm²) or the energy density (energy per unit area, in J/cm², orpower density multiplied by the exposure time) of the light energydelivered to tissue is an important factor in determining the relativeefficacy of the phototherapy, and efficacy is not as directly related tothe total power or the total energy delivered to the tissue. In themethods described herein, power density or energy density as deliveredto a portion of the patient's brain 20, which can include an areaaffected by neurodegenerative disease (e.g., Parkinson's disease),appears to be important factors in using phototherapy to treat the brain20. Certain embodiments apply optimal power densities or energydensities to the intended target tissue, within acceptable margins oferror.

As described in U.S. Patent Application Publication Nos. 2004/0138727A1,now U.S. Pat. No. 7,303,578, 2007/0179570A1, and 2007/0179571A1, nowU.S. Pat. No. 7,575,589, each of which is incorporated in its entiretyby reference herein, LLLT has been is particularly applicable withrespect to treating and saving surviving but endangered neurons afterstroke (e.g., in a zone of danger surrounding the primary infarct aftera stroke or cerebrovascular accident). In some embodiments light energydelivered within a certain range of power densities and energy densitiesprovides the desired biostimulative (or other biological) effect on theintracellular environment, such that proper function is returned topreviously nonfunctioning or poorly functioning mitochondria in neuronswhich are at risk due to stroke. The biological effect may includeinteractions with chromophores within the target tissue, whichfacilitate production of ATP thereby feeding energy to injured cellswhich have experienced decreased blood flow due to the stroke. Becausestrokes correspond to blockages or other interruptions of blood flow toportions of the brain, effects of increasing blood flow of said blockedvessels by phototherapy, in some embodiments, may be of less importancein the efficacy of phototherapy for stroke victims. In otherembodiments, treating vessels with interrupted flow may be beneficial.Further information regarding the role of power density and exposuretime is described by Hans H. F. I. van Breugel and P. R. Dop Bär in“Power Density and Exposure Time of He—Ne Laser Irradiation Are MoreImportant Than Total Energy Dose in Photo-Biomodulation of HumanFibroblasts In Vitro,” Lasers in Surgery and Medicine, Volume 12, pp.528-537 (1992), which is incorporated in its entirety by referenceherein.

A prominent feature of early Parkinson's disease is the damage to theneuronal processes (e.g., axons and their synapses) that communicatewith other neurons. Axons are thin, cylindrical processes that extend sofar from the neuronal cell that they require an axonal transport systemto supply vital nutrients and important organelles like mitochondria andsynaptic vesicles. One recent hypothesis to explain why axons andsynapses are damaged in Parkinson's disease patients is a failure in theaxonal transport system in dopaminergic neurons.

To determine if axonal transport is defective, two different models ofsporadic Parkinson's disease have been previously used in studies by Dr.Patricia Trimmer et al. of the University of Virginia Department ofNeuroscience. In these studies, axonal transport of mitochondria wasfound to be significantly reduced in processes of Parkinson's diseasecybrids (unique human neuronal cell lines that contain the mitochondrialDNA of individual Parkinson's disease patients and which share manyimportant attributes with injured dopaminergic neurons in the brains ofParkinson's disease patients) and similar human neuronal cells exposedto rotenone (a pesticide that damages neurons in a manner that resemblesParkinson's disease). These findings suggest that reduced axonaltransport plays an important role in the early stages of Parkinson'sdisease.

Studies which have exposed Parkinson's disease cybrid cells androtenone-treated neuronal cells to low energy laser treatment have foundthat axonal transport of mitochondria was restored. Such studiesillustrate that low energy laser treatment can improve the supply ofvital nutrients and organelles to axons and synapses in Parkinson'sdisease to compensate at least in part for the reduced axonal transport.In view of the hypothesis that axonal transport of essential nutrientsis reduced in Parkinson's disease, certain embodiments described hereinadvantageously provide low energy laser treatment to combat thisreduction of transport. In certain embodiments described herein,delivering electromagnetic radiation to brain cells causes animprovement of mitochondrial function in irradiated neurons.

In certain embodiments, the apparatus and methods of phototherapydescribed herein increase the cerebral blood flow of the patient. Incertain such embodiments, the cerebral blood flow is increased by atleast about 5%, 10%, 15%, 20%, or 25% immediately post-irradiation, ascompared to immediately prior to irradiation.

A number of studies have investigated the effects of in vitroirradiation of cells using pulsed light on various aspects of the cells.A study of the action mechanisms of incoherent pulsed radiation at awavelength of 820 nanometers (pulse repetition frequency of 10 Hz, pulsewidth of 20 milliseconds, dark period between pulses of 80 milliseconds,and duty factor (pulse duration to pulse period ratio) of 20%) on invitro cellular adhesion has found that pulsed infrared radiation at 820nanometers increases the cell-matrix attachment. (T. I. Karu et al.,“Cell Attachment to Extracellular Matrices is Modulated by PulsedRadiation at 820 nm and Chemicals that Modify the Activity of Enzymes inthe Plasma Membrane,” Lasers in Surgery and Medicine, Vol. 29, pp.274-281 (2001) which is incorporated in its entirety by referenceherein.) It was hypothesized in this study that the modulation of themonovalent ion fluxes through the plasma membrane, and not the releaseof arachidonic acid, is involved in the cellular signaling pathwaysactivated by irradiation at 820 nanometers. A study of light-inducedchanges to the membrane conductance of ventral photoreceptor cells foundbehavior which was dependent on the pulse parameters, indicative of twolight-induced membrane processes. (J. E. Lisman et al., “TwoLight-Induced Processes in the Photoreceptor Cells of Limulus VentralEye,” J. Gen. Physiology, Vol. 58, pp. 544-561 (1971), which isincorporated in its entirety by reference herein.) Studies oflaser-activated electron injection into oxidized cytochrome c oxidaseobserved kinetics which establish the reaction sequence of the protonpump mechanism and some of its thermodynamic properties have timeconstants on the order of a few milliseconds. (I. Belevich et al.,“Exploring the proton pump mechanism of cytochrome c oxidase in realtime,” Proc. Nat'l Acad. Sci., Vol. 104, pp. 2685-2690 (2007); I.Belevich et al., “Proton-coupled electron transfer drives the protonpump of cytochrome c oxidase,” Nature, Vol. 440, pp. 829-832 (2006),both of which are incorporated in its entirety by reference herein.) Anin vivo study of neural activation based on pulsed infrared lightproposed a photo-thermal effect from transient tissue temperaturechanges resulting in direct or indirect activation of transmembrane ionchannels causing propagation of the action potential. (J. Wells et al.,“Biophysical mechanisms responsible for pulsed low-level laserexcitation of neural tissue,” Proc. SPIE, Vol. 6084, pp. 60840X (2006),which is incorporated in its entirety by reference herein.)

In certain embodiments, delivering the neuroprotective amount of lightenergy includes selecting a surface power density of the light energy atthe scalp 30 corresponding to the predetermined power density at thetarget area of the brain 20. As described above, light propagatingthrough tissue is scattered and absorbed by the tissue. Calculations ofthe power density to be applied to the scalp 30 so as to deliver apredetermined power density to the selected target area of the brain 20preferably take into account the attenuation of the light energy as itpropagates through the skin and other tissues, such as bone and braintissue. Factors known to affect the attenuation of light propagating tothe brain 20 from the scalp 30 include, but are not limited to, skinpigmentation, the presence and color of hair over the area to betreated, amount of fat tissue, the presence of bruised tissue, skullthickness, and the location of the target area of the brain 20,particularly the depth of the area relative to the surface of the scalp30. For example, to obtain a desired power density of approximately 50mW/cm² in the brain 20 at a depth of 3 cm below the surface of the scalp30, phototherapy may utilize an applied power density of approximately3500 mW/cm². The higher the level of skin pigmentation, the higher thepower density applied to the scalp 30 to deliver a predetermined powerdensity of light energy to a subsurface site of the brain 20. Asdiscussed above, in some embodiments, blanching of the scalp (or otherpatient surface) defines, in part, the amount of light emissionnecessary to achieve a desired irradiance at the target tissue.

In certain embodiments, treating a patient suffering from the effects ofneurodegenerative disease (e.g., Parkinson's disease) comprises placingthe therapy apparatus 10 in contact with the scalp 30 and adjacent thetarget area of the patient's brain 20. The target area of the patient'sbrain 20 can be previously identified such as by using standard medicalimaging techniques. In certain embodiments, treatment further includescalculating a surface power density at the scalp 30 which corresponds toa preselected power density at the target area of the patient's brain20. The calculation of certain embodiments includes factors that affectthe penetration of the light energy and thus the power density at thetarget area. These factors include, but are not limited to, thethickness of the patient's skull, type of hair and hair coloration, skincoloration and pigmentation, patient's age, patient's gender, and thedistance to the target area within the brain 20. The power density andother parameters of the applied light are then adjusted according to theresults of the calculation.

The power density selected to be applied to the target area of thepatient's brain 20 depends on a number of factors, including, but notlimited to, the wavelength of the applied light, the type of CVA(ischemic or hemorrhagic), and the patient's clinical condition,including the extent of the affected brain area. The power density oflight energy to be delivered to the target area of the patient's brain(or other tissue) may also be adjusted to be combined with any othertherapeutic agent or agents, especially pharmaceutical neuroprotectiveagents (or for example, stem cell mobilizing compounds), to achieve thedesired biological effect. In such embodiments, the selected powerdensity can also depend on the additional therapeutic agent or agentschosen.

In certain embodiments, the treatment per treatment site proceedscontinuously for a period of about 10 seconds to about 2 hours, morepreferably for a period of about 1 to about 10 minutes, and mostpreferably for a period of about 1 to 5 minutes. For example, thetreatment time per treatment site in certain embodiments is about twominutes. In other embodiments, the light energy is preferably deliveredfor at least one treatment period of at least about five minutes, andmore preferably for at least one treatment period of at least tenminutes. The minimum treatment time of certain embodiments is limited bythe biological response time (which is on the order of microseconds).The maximum treatment time of certain embodiments is limited by heatingand by practical treatment times. The light energy can be pulsed duringthe treatment period or the light energy can be continuously appliedduring the treatment period.

In certain embodiments, the treatment may be terminated after onetreatment period, while in other embodiments, the treatment may berepeated for at least two treatment periods. The time between subsequenttreatment periods is preferably at least about five minutes, morepreferably at least about 1 to 2 days, and most preferably at leastabout one week. In certain embodiments in which treatment is performedover the course of multiple days, the apparatus 10 is wearable overmultiple concurrent days (e.g., embodiments of FIGS. 1, 3, 9A, 10, 13,and others disclosed, though not expressly depicted). The length oftreatment time and frequency of treatment periods can depend on severalfactors, including the functional recovery of the patient and theresults of imaging analysis of the infarct. In certain embodiments, oneor more treatment parameters can be adjusted in response to a feedbacksignal from a device (e.g., magnetic resonance imaging) monitoring thepatient.

During the treatment, the light energy may be continuously provided, orit may be pulsed. If the light is pulsed, the pulses are preferablyrange, in some embodiments from at least about 10 nanoseconds long toabout 50 milliseconds long, including about 10-100 ns, 100-500 ns, 500ns-1 ms, 1 ms-5 ms, 5-10 ms, 10-15 ms, 15-20 ms, 20-30 ms, 30-40 ms,40-50 ms, and occur overlapping ranges thereof. In some embodiments,pulses are administered for 1, 1.5, 2, 2.5, 3, 3.5, 4 or 4.5milliseconds. In some embodiments, pulses are administered for longerthan 50 milliseconds (e.g., 100 ms, 250 ms, 500 ms, 1 s, or higher).Pulsed light is administered, in some embodiments at a frequency of upto about 100 kHz. In several embodiments, lower frequencies are used,such as, for example, frequencies ranging from 50-150 Hz. In someembodiments, pulsed light is administered at about 60, 70, 80, 90, 95,100, 105, 110, 115, 120, 130, and 140 Hz. Frequencies less than 50 Hzand greater than 150 Hz are used in some embodiments. For example, inseveral embodiments, frequencies that match endogenous neuralfrequencies (e.g., Alpha, Beta, Delta, and/or Theta waves) are used. Insome embodiments pulsed light administration is preferred because of areduction in the amount of heat generated in the target tissue.Parameters may be chosen, in some embodiments to minimize heat. However,certain embodiments are particularly unexpected because the parametersused to generate the most robust effects are not the same as those thatwould minimize heat generation. As such, certain such embodiments maymore specifically target and affect a biological system (e.g. stem cellengraftment or proliferation) as compared to those parameters used tominimize heat.

In some embodiments, pulses described herein are administered in anon/off cycle (e.g., a duty cycle). In some embodiments, the duty cycleis between 0.01% to about 99.9% (e.g., between about 0.01%-0.1%,0.1%-1%, 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%,70%-80%, 80%-90%, 90%-99.9%, and overlapping ranges thereof). In oneembodiment, the on time is 2 ms and the off time is 1-2 ms. In anotherembodiment, the on time is about 1-5 ms and the off time is about 1-5ms. In some embodiments, the on/off times are variable during the courseof treatment. For example, in one embodiment, the on or off times areincreased (or decreased) by about 10-50% during the course of treatment.

In several embodiments, the duty cycle is synchronized with naturalneuronal rhythms. Mammalian neurons generate wave patterns of neuronalfiring that can be detected and measured by electroencephalography. Theprimary types of neuronal waves that have been detected are Alpha, Beta,Delta, and Theta waves.

Alpha waves occur in a frequency range of 8-13 Hz and are associatedwith states of low levels of activity or non-arousal. For example, aftercompleting a task and taking a period of rest, alpha waves may begenerated. Alpha waves are also associated with meditative states. Thus,in several embodiments synchronizing the duty cycle with alpha wavesenhances the normal effects associated with generation of alpha waves,e.g., relaxation, deeper thought etc.

Beta waves occur at frequencies ranging from about 13-40 Hz areassociated with higher levels of arousal and active engagement in mentalactivities. In several embodiments, synchronizing the duty cycle withbeta waves enhances the ability of an individual performing tasksassociated with beta wave generation. For example, in some embodiments,LLLT synchronized with beta waves enables longer periods ofconcentration, enhanced mental acuity, reduced fatigue after periods ofmental activity, etc.

Delta waves occur at frequencies ranging from about 1-4 Hz, the slowestfrequency of the various brain waves. Deep sleep commonly generatesDelta waves. In several embodiments, LLLT synchronized with delta wavesgeneration enhances the depth and/or perceived quality of sleep and/ordeep relaxation. In some embodiments, LLLT is used as a sleep aid, suchas for insomniacs, light sleepers, or those who have difficulty sleepingthrough the night. In some embodiments, LLLT can be used to enhancesleep sessions of those individuals having uncommon or variable workhours (e.g., work at night and sleep during the day).

Theta waves occur at frequencies ranging from about 4-7 Hz. Theta wavesmay be generated when a person is aware of his/her surroundings butdaydreaming or otherwise not focusing on any task in particular. Is somecases, theta waves are associated with free flow of thought andgeneration of creative ideas. In several embodiments, LLLT synchronizedwith theta waves enhances an individual's creative thought processenables an individual to generate new ideas and/or thoughts. Suchembodiments can be used to, among other applications, assist inovercoming mental blocks (e.g., writer's block or phobias), enhance theefficiency of brainstorming sessions, and/or assist individuals orgroups in problem solving.

In some embodiments, the duty cycle is selected to reflect cellularrefractory periods (e.g., the refractory period of a cardiac cell).

The course of the action potential in excitable cells comprises fiveparts: the rising phase, the peak phase, the falling phase, theundershoot phase, and finally the refractory period. During the risingphase the membrane potential depolarizes (becomes more positive,typically from a resting potential of about −70 mV), due to opening ofvoltage-gated sodium ion channels open, which increases membraneconductance for sodium ions. Once the membrane potential reaches adepolarization threshold (about −35 to about −40 mV) the opening ofsodium channels will cause other sodium channels open, resulting in afeed-forward rapid depolarization. The point at which depolarizationstops is called the peak phase. At this stage, the membrane potentialreaches a maximum. Subsequent to this, there is a falling phase. Duringthis stage the membrane potential hyperpolarizes (becomes morenegative). During repolarization, voltage-gated sodium ion channelsinactivate and voltage-gated potassium channels activate. Both thesodium ion channels closing and the potassium ion channels opening actto repolarize the cell's membrane potential back towards the restingmembrane potential.

However, the potassium conductance has a lag time that leads to a shorthyperpolarization, known as the undershoot phase. This period ofhyperpolarization is known as the refractory period. Eventually thispotassium conductance drops and the exits the refractory period and cellreturns to its resting membrane potential.

There are two refractory periods in excitable cells (e.g., neurons). Theabsolute refractory period is the time period after a first stimulationduring which a second stimulation of the cell will not trigger an actionpotential (or other cellular response normally associated with astimulus). The absolute refractory period of neurons typically rangefrom about 1 to about 3 milliseconds. Thus, in several embodiments, theduty cycle is adjusted to provide light administration to the cells(e.g., neurons) approximately every 1-3 milliseconds, or in sync withthe absolute refractory period

The relative refractory period is the time period after a firststimulation during which the probability of a second stimulation of thecell triggering an action potential (or other cellular response normallyassociated with a stimulus) is reduced, but an action potential maystill be possible. The relative refractory period immediately followsthe absolute refractory period. During the relative refractory period, astimulus will need to be proportionally greater (to account for thehyperpolarization) in order to cause the membrane potential of the cellto reach the depolarization threshold, and initiate a new actionpotential. Absent an additional stimulus, the potassium conductance willreturn to its resting value and the membrane potential of the cell willreturn to equilibrium, thus ending the relative refractory period.

As the refractory period is varied depending on the cell type, greateror lesser refractory periods can be accommodated by adjusting the dutycycle. For example, in some embodiments, the duty cycle is adjusted toprovide light to the cell approximately every 0.8-1.0 seconds, 1.0-1.2seconds, 1.2-1.4 seconds, 1.4-1.6 seconds, 1.6-1.8 seconds, 1.8-2.0seconds, 2.0-2.2 seconds, 2.2-2.4 seconds, 2.4-2.6 seconds, 2.6-2.8seconds, and 2.8-3.0 seconds (and overlapping ranges thereof).Synchronization of LLLT, in some embodiments, enhances the function ofthe exposed cells. For example, synchronizing light administration withthe refractory period of a sensory neuron, in some embodiments,increases the rate of sensory transmission in the neuron, which, in someembodiments, produces heightened sensory capacity. Additionally,synchronization of LLLT with the refractory period of motor neurons, insome embodiments, aids in normalization of neuronal firing rates,thereby increasing fine motor control and/or serving as a therapy orpalsies or other such uncontrolled muscle movements.

In some embodiments, LLLT, whether continuous or pulsed, is administeredfor a total time (duration per treatment session at one site) of about 1second to 10 minutes, e.g., between about 1 s to 25 s, 25 s-50 s, 50s-100 s, 1 minute-2 minutes, 2 minutes-3 minutes, 3 minutes-4 minutes, 4minutes-5 minutes, 5 minutes-6 minutes, 6 minutes-7 minutes, 7 minutes-8minutes, 8 minutes-9 minutes, 9 minutes-10 minutes, or greater. In someembodiments, the total time (duration per treatment session at one site)is about 40, 50, 60, 70, 60, 90 100, 110, 120 seconds. In severalembodiments, treatment is performed on one or more sites (e.g., 2, 3, 4,5, 10, 15, 20, 25, 30 or more sites). In several embodiments, multipletreatment sessions are performed at different times (e.g., differenthours, different days, etc.) on the same site (or at different sites).

In one embodiment, the invention comprises delivering pulsed LLLT to aneuron (or group neurons) every 1-2 milliseconds. In one embodiment, theinvention comprises delivering pulsed LLLT to a cell (e.g., an excitablecell such as a neuron) in synchronicity with the activation ordeactivation of an ion channel (e.g., sodium, calcium or potassiumchannel). In some embodiments, the LLLT is administered before an actionpotential occurs. In several embodiments, LLLT is administered in syncwith the depolarization phase of the action potential. In severalembodiments, LLLT is administered in sync with the peak phase of theaction potential. In several embodiments, LLLT is administered in syncwith the repolarization phase of the action potential. In severalembodiments, LLLT is administered in sync with the hyperpolarizationphase of the action potential. In some embodiments, the LLLT isadministered during the relative refractory period, while in someembodiments, the LLLT is administered during the relative refractoryperiod. In several embodiments, LLLT is administered for a period oftime that overlaps one or more phases of an action potential. In severalembodiments, LLLT is administered in sync, preceding, or following aparticular action potential event. For example, in some embodiments,LLLT is administered based on the opening of sodium channels, while insome embodiments, LLLT is administered based on the potassium inducedhyperpolarization of the cell membrane.

In certain embodiments, the treatment per treatment site proceedscontinuously for a period of about 10 seconds to about 2 hours, for aperiod of about 1 to about 10 minutes, or for a period of about 1 to 5minutes. For example, the treatment time per treatment site in certainembodiments is about two minutes. In other embodiments, the light energyis delivered for at least one treatment period of at least about fiveminutes, or for at least one treatment period of at least ten minutes.The minimum treatment time of certain embodiments is limited by thebiological response time (which is on the order of microseconds). Themaximum treatment time of certain embodiments is limited by heating andby practical treatment times (e.g., completing treatment prior to orbetween other treatment regimens). The light energy can be pulsed duringthe treatment period or the light energy can be continuously appliedduring the treatment period. If the light is pulsed, the pulses can be 2milliseconds long and occur at a frequency of 100 Hz, although longerpulselengths and lower frequencies can be used, or at least about 10nanosecond long and occur at a frequency of up to about 100 kHz.

In certain embodiments, the treatment may be terminated after onetreatment period, while in other embodiments, the treatment may berepeated for at least two treatment periods. The time between subsequenttreatment periods can be at least about five minutes, at least two in a24-hour period, at least about 1 to 2 days, or at least about one week.The length of treatment time and frequency of treatment periods candepend on several factors, including the functional recovery of thepatient. In certain embodiments, one or more treatment parameters can beadjusted in response to a feedback signal from a device (e.g., magneticresonance imaging) monitoring the patient.

In addition to the combination of cell therapy and phototherapy, incertain embodiments, the phototherapy is combined with other types oftreatments for an improved therapeutic effect. Treatment can comprisedirecting light through the scalp of the patient to a target area of thebrain concurrently with applying an electromagnetic field to the brain.Similar approaches are taken to treat other target tissues. In suchembodiments, the light has an efficacious power density at the targetarea and the electromagnetic field has an efficacious field strength.For example, the apparatus 50 can also include systems forelectromagnetic treatment, e.g., as described in U.S. Pat. No. 6,042,531issued to Holcomb, which is incorporated in its entirety by referenceherein. In certain embodiments, the electromagnetic field comprises amagnetic field, while in other embodiments, the electromagnetic fieldcomprises a radio-frequency (RF) field. As another example, treatmentcan comprise directing an efficacious power density of light through thescalp of the patient to a target area of the brain concurrently withapplying an efficacious amount of ultrasonic energy to the brain. Such asystem can include systems for ultrasonic treatment, e.g., as describedin U.S. Pat. No. 5,054,470 issued to Fry et al., which is incorporatedin its entirety by reference herein.

Assessing Efficacy of Light Therapy and Cell Therapy

Depending on the disease or injury treated by LLLT and cell therapy,various endpoints are used to assess the efficacy of the therapies. Forexample, neurologic function scales can be used to quantify or otherwisecharacterize the efficacy of various embodiments described herein.Neurologic function scales generally use a number of levels or points,each point corresponding to an aspect of the patient's condition. Thenumber of points for a patient can be used to quantify the patient'scondition, and improvements in the patient's condition can be expressedby changes of the number of points. One example neurologic functionscale used as a clinical tool for diagnosis and determining severity ofParkinson's disease is the Unified Parkinson's Disease Rating Scale(UPDRS) which comprises various sections evaluated by interview andclinical observation. In certain embodiments, two or more of theneurologic function scales can be used in combination with one another,and can provide longer-term measurements of efficacy (e.g., at threemonths).

In certain embodiments described herein, a patient exhibiting symptomsof Parkinson's disease is treated by irradiating a plurality oftreatment sites on the patient's scalp. The irradiation is performedutilizing irradiation parameters (e.g., wavelength, power density, timeperiod of irradiation, etc.) which, when applied to members of a treatedgroup of patients, produce at least a 2% average difference between thetreated group and a placebo group on at least one neurologic functionscale (e.g., UPDRS) analyzed in dichotomized or any other fashion.Certain other embodiments produce at least a 4% average difference, atleast a 6% average difference, or at least a 10% average differencebetween treated and placebo groups on at least one neurologic functionscale analyzed in dichotomized or any other fashion. In certainembodiments, the irradiation of the patient's scalp produces a change inthe patient's condition. In certain such embodiments, the change in thepatient's condition corresponds to a change in the number of pointsindicative of the patient's condition. In certain such embodiments, theirradiation produces a change of one point, a change of two points, achange of three points, or a change of more than three points on aneurologic function scale.

Other diseases or injuries that are treated with LLLT and cell therapycan be assessed by standard clinical measures related to that disease orinjury. For example, treatment of cardiac tissue damage after MI can beevaluated by laboratory measurements of total cardiac output,echocardiography to measure ventricular function, or stress testing tomeasure an individual's overall cardiovascular performance. Effects ofadministration of a stem cell mobilizing compound and LLLT irradiationof bone marrow can be measured by a complete blood count.

Possible Action Mechanisms

The following section discusses theories and potential actionmechanisms, as they presently appear to the inventors, for certainembodiments of phototherapy described herein. The scope of the claims ofthe present application is not to be construed to depend on theaccuracy, relevance, or specifics of any of these theories or potentialaction mechanisms. Thus the claims of the present application are to beconstrued without being bound by theory or by a specific mechanism.

There are five large components of the electron transfer chain,Complexes I-IV and the ATPase (also called Complex V), with each complexcontaining a number of individual proteins (see FIG. 34). One of thecritical complexes, Complex IV (cytochrome oxidase), is the componentresponsible for the metabolism of oxygen. The cytochrome C oxidaseprotein is a key player in the electron transfer in Complex IV throughits copper centers. In several embodiments, administered light isabsorbed (photoaccepted) by one or more of these copper centers.

As an aside, cytochrome C oxidase has enjoyed a renaissance in the lastfew years as an important factor in the regulation of apoptosis(programmed cell death). Release of cytochrome C oxidase from themitochondria into the cytosol is a pro-apoptotic signal.

Light is known to affect biological systems, such as vision, regulationof circadian hormones, melanin production, and Vitamin D synthesis. Withrespect to specific organelles, specific wavelengths of light targetedat the cytochrome C receptor in the mitochondria can preservemitochondrial function, as well as, reduce the size of myocardialinfarcts and stroke. It has also been shown that these effects can bereproduced across multiple species. Given that there is someconservation of the mitochondrial target receptor (i.e., copper ions incytochrome C) between species, effects demonstrated in animal models arelikely to be readily translated to similar effects in human patients.These effects may be due to production of new cells (neurogenesis),preservation of existing tissue (neuroprotection) or a combination ofboth.

The mitochondria convert oxygen and a carbon source to water and carbondioxide, producing energy (as ATP) and reducing equivalents (redoxstate) in the process. The process details of the electron transportchain in mitochondria are schematically diagrammed in FIG. 34. Thechemical energy released from glucose and oxygen is converted to aproton gradient across the inner membrane of the mitochondria. Thisgradient is, in turn, used by the ATPase complex to make ATP. Inaddition, the flow of electrons down the electron transfer chainproduces NADPH and NADH (and other factors such as FAD). These cofactorsare important for maintaining the redox potential inside the cell withinthe optimal range. This process has been called the chemi-osmotic theoryof mitochondrial function (Dr. Peter Mitchell was awarded a Nobel Prizein chemistry for elucidating these key processes).

The clinical and cellular responses to light for in vivo treatmentefficacy of ischemic conditions of acute myocardial infarction andstroke has been demonstrated in multiple validated animal models. Asdescribed more herein, these effects are wavelength-specific. In severalembodiments, the wavelength specificity may be dependent upon a knownmitochondrial receptor (cyctochrome C oxidase). In some embodiments,targeting of this receptor results in formation of adenosinetriphosphate (ATP), enhanced mitochondrial survival, and/or maintenanceof cytochrome C oxidase activity.

In stroke, the occlusion of a major artery results in a core area ofsevere ischemia (e.g., with significant reductions in blood flow, forexample flow reduced to less than 20% of pre-occlusion levels). The corearea has a rapid loss of ATP and energy production, and the neurons aredepolarized. This core of the infarct is surrounded by an ischemicpenumbra which can be up to twice as large as the core of the infarct.Cells within the penumbra show less severe decreases in loss of bloodflow (e.g., ranging up to 20 to 40%, or more, of normal). Neurons in thepenumbra tend to be hyperpolarized and electrically silent. In thepenumbra, the cells undergo progression of cell death lasting from hoursto days after the infarct. Also, inflammation after infarct can play arole in determining the final infarct size and anti-inflammatorymodulators can reduce infarct size. The infarct is dynamic, withdifferent parts of the infarct being affected to different degrees overa period of hours to days. Photon therapy has been implicated in anumber of physiological processes that could favor cell survival in thepenumbral region of a stroke.

The action of light on a cell is mediated by one or more specific photoacceptors. A photo acceptor molecule first absorbs the light. After thisabsorptive event and promotion of an electron to an excited state, oneor more primary molecular processes from these high energy states canlead to a measurable biological effect at the cellular level. An actionspectra represents the biological activity as a function of wavelength,frequency or photon energy. Karu was the first researcher to proposethat the action spectra should resemble the absorption spectra of thephotoacceptor molecule. Since an absorptive event occurs for a transferof energy to take place, the stimulatory wavelengths of the actionspectra falls within the absorptive spectra of the photo acceptor.

It has been postulated by others that light can directly activateComplex IV and indirectly driving the production of ATP via ATPase (andreducing equivalents). For example, Karu studied the activation spectraof these processes and found that wavelengths that maximally stimulatedenergy-dependant cellular functions corresponded to the absorption bandsof the copper centers in cytochrome C oxidase. FIG. 36 is a graph ofcell proliferation and cytochrome oxidase activity percentage asfunctions of the wavelength of light used to stimulate mammalian cells.Based on these results, wavelengths of 620, 680, 760, and 820 nanometers(±10 nanometers) promote cellular activities. The 620 and 820 nanometerwavelengths are close to the strongest copper absorption maxima of 635and 810 nanometers.

Karu was also the first to propose a specific mechanism for photontherapy at the cellular level (see, e.g., T. Karu, “PhotobiologicalFundamentals of Low Power Laser Therapy,” IEEE Journal of QuantumElectronics, 1987, Vol. 23, page 1703; T. Karu, “Mechanisms ofinteraction of monochromatic visible light with cells,” Proc. SPIE,1995, Vol. 2630, pages 2-9). Karu's hypothesis was based on theabsorption of monochromatic visible and near infrared radiation bycomponents of the cellular respiratory chain. Absorption and promotionof electronically excited states cause changes in redox properties ofthese molecules and acceleration of electron transfer (primaryreactions). Primary reactions in mitochondria of eukaryotic cells arefollowed by a cascade of secondary reactions occurring in the cytoplasm,cell membrane, and nucleus. Karu defined the action spectra formammalian cells of several secondary reactions (DNA, RNA synthesis,cellular adhesion). The action spectra for all of these secondarymarkers were very similar, suggesting a common photo acceptor. Karu thencompared these action spectra with absorption spectra of the coppercenters of cytochrome C oxidase in both reduced and oxidized states.Cytochrome C oxidase contains four redox active metal centers and has astrong absorbance in the near infrared spectral range. The spectralabsorbance of cytochrome C oxidase and the action spectra were verysimilar. Based on this, Karu suggested that the primary photoacceptorsare mixed valence copper centers within cytochrome C oxidase.

Cytochrome C oxidase is the terminal enzyme of the mitochondrialelectron transport chain of all eukaryotes and is required for theproper function of almost all cells, especially those of highlymetabolically active organs, such as the brain and heart. Cytochrome Chas also been suggested to be the critical chromophore responsible forstimulatory effects of irradiation with infrared light to reverse thereduction in cytochrome C oxidase activity produced by the blockade ofvoltage dependent sodium channels with tetrodotoxin and up regulatedcytochrome C activity in primary neuronal cells. It has beendemonstrated by researchers (see, e.g., M. T. Wong-Riley et al.,NeuroReport, 2001, Vol. 12, pages 3033-3037; J. T. Eells et al.,Proceedings National Academy of Science, 2003, Vol. 100, pages3439-3444) that in vivo, rat retinal neurons are protected from damageinduced by methanol intoxication. Methanol's toxic metabolite is formicacid which inhibits cytochrome C.

Several investigators have demonstrated the increased synthesis of ATPfrom infrared irradiation both in vitro and in vivo. Karu has shown thatirradiation of cells in vitro at wavelengths of 632 nanometers, 670nanometers, and 820 nanometers can increase mitochondrial activity.

Additional data from other groups suggest that cytochrome C oxidase isan important target. Light (670 nanometers) can rescue primary neuronsfrom the toxic effects of the sodium channel blocker tetrodotoxin (TTX).TTX reduces cytochrome oxidase activity in treated neurons, and thisreduction is reversed by light treatment (an increase in cytochromeoxidase activity). In an in vivo model, 670 nanometer light is used torescue retinal function in a methanol-mediated model of retinal damage.Methanol is metabolized to formate, a selective mitochondrial toxintargeted at cytochrome C oxidase. Irradiation with light (670nanometers) rescued the retina from damage induced by methanol.

Several studies hypothesize that photon therapy would be effective inanimal models for acute myocardial infarction (AMI) and ischemic stroke,by virtue of the photon therapy inducing a cascade of signaling eventsinitiated by the initial absorption of light by cytochrome C. Thesesignaling events apparently up-and-down regulate genes, transcriptionfactors, as well as increase mitochondrial function.

Without being bound by theory or a specific mechanism, in stroke,reduction of infarct volume may occur in one of two ways or acombination of both: (i) preservation of existing tissue(neuroprotection), and (ii) generation of new tissue (neurogenesis). Anumber in vitro and in vivo studies appear to support both of thesepotential mechanisms. The potential effects of NIR light on neurogenesisare straight-forward; it either increases the number of new cells, or itprevents the loss of new cells that are generated as a result of theischemic insult. Neuroprotection can result from at least threemechanisms: (i) direct stimulation of tissue survival; (ii) indirectstimulation of tissue survival (e.g., increased growth factor activity);and (iii) decrease in toxic factors. Analogous mechanisms are likelyinvolved in the survival of other tissues in response to injury (e.g.,cardioprotective mechanism).

FIG. 35 is a graph which shows mediators responsible for ischemic stroketissue damage and the time points at which they occur. FIG. 35illustrates several potential places where photon therapy couldpotentially intervene to reduce infarct severity. Early after ischemicstroke, excitatory amino acids (EAAs) induce Ca²⁺ influx via NMDAreceptor activation leading to neuronal and glial cell injury. A numberof immediately early genes (IEGs) express such as c-fos, c-jun, within30 minutes. Reactive oxygen species (ROSs) create lipid peroxidation andactivated phagocytes which create further injury. ROSs damage mostcellular components. Cytokines are then expressed causing migration ofpolymorphonuclear neutrophils (PMNs) into the ischemic brain.Macrophages and neutrophils follow into the brain parenchyma. Apoptosisoccurs via caspase activation which further increases stroke damage.

Preservation of existing tissue (neuroprotection) can result from directstimulation of the tissue (e.g., by ATP synthesis or by prevention ofcytochrome C release from mitochondria). Ischemia results in depletionof ATP in the ischemic zone due to lack of oxygen and glucose. Theresultant lack of ATP, depending on severity, results in decreasedcellular function. In extreme cases, energy depletion leads to celldepolarization, calcium influx, and activation of necrotic and apoptoticprocesses. Near-infrared radiation (NIR) stimulates the production ofATP in a variety of cell types in culture, and in cardiac tissue. Asingle irradiation of infarcted cardiac tissue results in astatistically significant 3-fold increase in tissue ATP levels fourhours after treatment. The effect of NIR is prolonged long afterirradiation is ceased. The prolonged effect could also be due, in part,to preservation of mitochondrial function. NIR irradiated, infarctedcardiac tissue has exhibited over a 50% reduction in damagedmitochondria. After ischemia, the myocardial tissue that is notimmediately lost is in a “stunned” state, and can remain stunned for aperiod of days. In particular, it is the mitochondria in the tissue thatare stunned. Stunned mitochondria are still intact, but withcharacteristic morphological changes that are indicative of mitochondriathat are not metabolically active. As such, even with restored bloodflow, the mitochondria are unable to convert oxygen and glucose touseable energy (ATP).

Neuroprotection can also result from direct stimulation of the tissue bypreventing cytochrome C release from mitochondria. The release ofcytochrome C from the mitochondria into the cytoplasm is a potentapoptotic signal. Cytochrome C release results in the activation ofcaspase-3 and activation of apoptotic pathways. The apoptotic cellsappear as soon as a few hours after stroke, but the cell numbers peak at24 to 48 hours after reperfusion. In rat models of stroke, cytoplasmiccytochrome C can be detected out to at least 24 hours after theocclusion. In vitro 810-nanometer light can prevent the TTX-induceddecrease in cytochrome oxidase activity. In several embodiments, photontherapy maintains cytochrome oxidase activity in vivo by preventingrelease of cytochrome C into the cytoplasm, resulting in the preventionof apoptosis. The release of cytochrome C is regulated by the Bcl/Baxsystem. Bax promotes release and Bcl decreases release. In myofibercultures in vitro, NIR light promotes Bcl-2 expression and inhibits Baxexpression, which fits with the prevention of cytochrome C release data.Thus, in some embodiments, light therapy has an effect on one or morelevels of the biochemical cascade that controls neuronal (or cellular)viability.

Neuroprotection can also result from indirect stimulation (e.g., byangiogenesis or by up-regulation of cell survival genes and/or growthfactors). Regarding angiogenesis and stroke, recent research indicatesthat the reduction in cerebral blood flow (CBF) can lead to compensatoryneovascularization in the affected regions. The low CBF results in theup regulation of hypoxia inducible factor-1 (HIF-1), vascularendothelial growth factor (VEGF), and VEGF receptors. In the rat pMCAomodel, infusion of VEGF results in a reduction of infarct size. In AMImodels, VEGF is increased with photon therapy.

Regarding up-regulation of cell survival genes and/or growth factors, ithas been shown that photon therapy may up-, and down-regulate certainbeneficial genes. It is possible that these gene products can prevent orameliorate apoptosis, which is known to occur throughout the stokepenumbra and in stunned myocardium of AMI. In AMI models, expression ofthe cardioprotective molecules HSP70 and VEGF are increased. In stroke,equivalent neuroprotective molecules could be up-regulated, preservingtissue and resulting in reduction of infarct volumes. A variety offactors have been implicated in neuroprotection in addition to VEGF,including BDNF, GDNF, EGF, FGF, NT-3, etc. In several embodiments, oneor more of these of factors that are up-regulated to promote neuronal(or other cellular) survival. In some embodiments, LLLT increases one ormore of the following: mitochondrial respiration, production ofmolecular oxygen, DNA synthesis, DNA repair, and cell proliferation. Incertain embodiments, these factors play a role in enhancing theviability, proliferation, migration, and/or engraftment of administeredstem cells.

Neuroprotection can also result from decreases in toxic factors (e.g.,antioxidant protection or by reduction of deleterious factors to tissuefunction and survival). Regarding antioxidant protection, in someembodiments, NIR light may reduce damage induced by free radicals.By-products of free radical damage are found in damaged brain tissuefollowing stroke. This damage is thought to be mediated by neutrophilsduring reperfusion injury. The nominal spin-trap agent NXY-059 (a freeradical scavenger) reduces infarct size if given within 2.25 hours of astroke (in rat, although it is more effective if given sooner). NIRlight can induce the expression of catalase in AMI models. Catalase is apowerful anti-oxidant which can prevent free radical damage and, ifproduced in the area of the stroke, it may prevent loss via the samemechanism as NXY-059. Axon survival is known to be improved by catalase.

In addition, a number of cytokines and other factors are produced duringreperfusion that are deleterious to tissue function and survival. Thesefactors promote activity of existing phagocytic and lymphocytic cells aswell as attract additional cells to the area of damage. In severalembodiments, NIR light can decrease the levels of cytokines in models ofneuronal damage. In particular, IL-6 and MCP-1 (pro-inflammatorycytokines) are induced in models of spinal cord damage. NIR lightsignificantly reduces IL-6 and MCP-1 and promotes regrowth of the spinalcords neurons. IL-6 is thought to play a significant role in spinal corddamage in man also.

Regarding neurogenesis, in the last several years, it has been becomewell-established that the brain has the ability to generate new nervecells in certain instances. Neural stem cells have been shown to existin the periventricular areas and in the hippocampus. Naturally-occurringgrowth factors in the adult human brain can spur the production of newnerve cells from these stem cells. After a stroke, neurogenesiscommences in the hippocampus with some cells actually migrating to thedamaged area and becoming adult neurons.

In several embodiments, NIR light is effective because administrationeither increases the number of new cells that are formed, or preventsthe loss of the newly formed cells. The latter may be more significantand the majority of newly-formed cells die within 2 to 5 weeks after thestroke (rat model). In an unpublished study by Oron, NIR light has beenshown to increase the survival of cardiomyocytes implanted intoinfarcted heart. Other studies have shown the human neural progenitorcells can be induced to differentiate with stimulation of 810-nanometerirradiation without the presence of specific growth factors that arenormally required for differentiation. In several embodiments,neurogenesis occurs due to the administered if the infrared irradiationacting as a stimulating signal much like a growth factor. Early datafrom a porcine study of AMI has shown that the 810-nanometer-irradiatedpig myocardium showed evidence of cardiogenesis. This result wasdemonstrated by the presence of significant desmin staining in the lasertreated group over control, and by ultrastructural analysis whichdemonstrated the presence of what appears to be developingcardiomyocytes.

In vitro and near in vitro like conditions (retinal studies) havepreviously demonstrated that light can induce beneficial effects inanimals. Yet these effects required little if any ability to penetratenon-involved tissues. For treatments of Parkinson's disease byirradiation through intervening tissue, certain embodiments utilizewavelengths that can penetrate to the target tissue.

Light can be absorbed by a variety of chromophores. Some chromophores,such as cytochrome C oxidase can convert the light energy into chemicalenergy for the cell. Other chromophores can be simple and the lightenergy is converted to heat, for example water. The absorption of lightenergy is wavelength dependent and chromophore dependant.

Some chromophores, such as water or hemoglobin, are ubiquitous andabsorb light to such a degree that little or no penetration of lightenergy into a tissue occurs. For example, water absorbs light aboveapproximately 1300 nanometers. Thus energy in this range has littleability to penetrate tissue due to the water content. However, water istransparent or nearly transparent in wavelengths between 300 and 1300nanometers. Another example is hemoglobin, which absorbs heavily in theregion between 300 and 670 nanometers, but is reasonably transparentabove 670 nanometers.

As discussed above, one can define an “IR window” into the body whichencompasses certain wavelengths that are more or less likely topenetrate. The absorption/transmittance of various tissues have beendirectly measured to determine the utility of various wavelengths.

FIG. 37 is a graph of the transmittance of light through blood (inarbitrary units) as a function of wavelength. Blood absorbs less in theregion above 700 nanometers, and is particularly transparent atwavelengths above 780 nanometers. Wavelengths below 700 nanometers areheavily absorbed, and are not likely to be useful therapeutically(except for topical indications). However, in certain embodiments,wavelengths below 700 nm are beneficial, for example in treating stemcells prior to delivery to a subject (e.g., as an in vitropre-treatment).

FIG. 38 is a graph of the absorption of light by brain tissue.Absorption in the brain is strong for wavelengths between 620 and 900nanometers. This range is also where the copper centers in mitochondriaabsorb. The brain is particularly rich in mitochondria as it is a veryactive tissue metabolically (the brain accounts for 20% of blood flowand oxygen consumption). As such, the absorption of light in the 620 to900 nanometer range is expected if a photostimulative effect is to takeplace. As discussed herein, in several embodiments, light isadministered to the brain (whole or portions thereof) at wavelengthsbetween about 620-900 nm.

By combining FIGS. 37 and 38, the efficiency of energy delivery as afunction of wavelength can be calculated, as shown in FIG. 39.Wavelengths between 780 and 880 nanometers are preferable (efficiency of0.6 or greater) for targeting the brain. The peak efficiency is about800 to 830 nanometers (efficiency of 1.0 or greater). These wavelengthsare not absorbed by water or hemoglobin, and are likely to penetrate tothe brain. Once these wavelengths reach the brain, they will be absorbedby the brain and converted to useful energy in several embodiments.

These effects have been directly demonstrated in rat tissues. Theabsorption of 808 nanometer light was measured through various rattissues, as shown in FIG. 40. Soft tissues such as skin and fat absorblittle light. Muscle, richer in mitochondria, absorbs more light. Evenbone is fairly transparent. However, as noted above, brain tissue, aswell as spinal cord tissue, absorb 808 nanometer light well.

Two wavelengths have demonstrated efficacy in animal models ofischemia/mitochondrial damage, namely, 670 nanometers and 808nanometers. Light having a wavelength of 670 nanometers has shownefficacy in retinal damage. Light having a wavelength of 808 nanometershas demonstrated efficacy in animal models of myocardial infarction (aswell as soft tissue injury).

The effects of near infrared light on soft tissue injury have beenestablished in FDA approved trials for carpal tunnel syndrome (830nanometers) and knee tendonitis (830 nanometers). In both cases, 830nanometer light was superior to placebo for resolution of symptoms.

Light having a wavelength of 808 nanometers was also used to reduceinfarct volume and mortality in myocardial infarction (MI) models inrat, dog, and pig. The MI models are particularly relevant to wavelengthselection as similar processes—apoptosis, calcium flux, mitochondrialdamage—have been implicated in stroke and MI.

Certain wavelengths of light are associated with activation ofbiological processes, and others are not. In particular, light mediatedmitochondrial activation has been used as a marker of biostimulation.Given the lack of in vivo markers, the use of in vitro markers of lightactivation was used to help narrow down the large number of potentialwavelengths. Wavelengths that activate mitochondria were determined, andthese wavelengths were used in vivo models.

Penetration to the target tissue is also of importance. If a biologicaleffect is to be stimulated, then the stimulus must reach the targettissue and cell. In this regard, wavelengths between 800 and 900nanometers are useful, as they can penetrate into the body. Inparticular, wavelengths of 800 to 830 nanometers are efficient atpenetrating to the brain and then being absorbed by the brain.

The use of 808 nanometer light has a solid basis for the treatment ofstroke. This wavelength of light can penetrate to the target tissue(brain), is absorbed by the target tissue, stimulates mitochondrialfunction, and works in a related animal model of ischemia (MI). Thissupposition is supported by the striking finding that 808 nanometerlight can reduce the neurological deficits and infarct volume associatedwith stroke (in rats).

Other wavelengths have some of these properties. For example, 670nanometer light can promote mitochondrial function and preserve retinalneurons. However, this wavelength does not penetrate tissue well as itis highly absorbed by hemoglobin. It is therefore not useful in treatingstroke or neurodegenerative conditions.

In certain embodiments, wavelengths from 630 to 904 nanometers may beused. This range includes the wavelengths that activate mitochondria invitro, and that have effects in animal models. These wavelengths alsoinclude the predominant bands that can penetrate into the body.

Transmission in Human Brain

Power density measurements have been made to determine the transmissionof laser light having a wavelength of approximately 808 nanometersthrough successive layers of human brain tissue. Laser light having awavelength of (808±5) nanometers with a maximum output of approximately35 Watts was applied to the surface of the cortex using a beam deliverysystem which approximated the beam profile after the laser light passesthrough the human skull. Peak power density measurements were takenthrough sections of human brain tissue using an Ocean Opticsspectrophotometer Model USB 2000, Serial No. G1965 and beam diameterafter scattering was approximated using a Sony Model DCR-IP220, SerialNo. 132289.

A fresh human brain and spinal cord specimen (obtained within six hoursafter death) was collected and placed in physiologic Dakins solution.The pia layer, arachnoid layer, and vasculature were intact. The brainwas sectioned in the midline sagittally and the section was placed in acontainer and measurements taken at thicknesses of 4.0 centimeters (±0.5centimeter), 2.5 centimeters (±0.3 centimeter), and 1.5 centimeters(±0.2 centimeter). The power density measurements are shown in Table 2:

TABLE 2 Thickness Power density at cortex Average power density atthickness 4.0 cm 20 mW/cm² 4.9 μW/cm²  2.5 cm 20 mW/cm² 20 μW/cm² 1.5 cm10 mW/cm² 148 μW/cm² 

FIG. 41 is a graph of the power density versus the depth from the durafor an input power density of 10 mW/cm² with the light barscorresponding to predicted values of the power density and dark barscorresponding to an estimated minimum working power density of 7.5μW/cm², as described below.

Based upon prior animal experimentation, a conservative estimation ofthe minimum known power density within the tissue of the brain which isable to show efficacy in stroke animal models is 7.5 μW/cm². Thisestimated minimum working power density is drawn from an experiment inwhich 10 mW was applied to the rat brain surface, and 7.5 μW/cm² powerdensity was directly measured 1.8 centimeters from the surface. Thisstroke model consistently produced significant efficacy, including forstrokes 1.8 centimeters from the laser probe. Note that this 7.5 μW/cm²is a conservative estimate; the same power density at the brain surfacealso consistently produces significant efficacy in the 3 centimeterrabbit clot shower model. Note also that the power density measurementsin the human brain experiment do not factor in the effect from theCNS-filled sulci, through which the laser energy should be readilytransmitted. However, even conservatively assuming 7.5 μW/cm² as theminimum power density hurdle and ignoring expected transmission benefitsfrom the sulci, the experiment described above confirms thatapproximately 10-15 mW/cm² transmitted upon the cortex (as per anexample dosimetry in man) will be effective to at least 3.0 centimetersfrom the surface of the brain.

In Vivo Thermal Measurements

In vivo thermal measurements were made to determine the heating effectin living tissue of laser light having a wavelength of approximately 808nanometers. A GaAlAs laser source of 808-nanometer light was placed indirect contact with the skin of the heads of live rabbits and rats. Thelaser source had an approximately Gaussian beam profile with a beamdiameter of 2.5-4.0 millimeters (1/e²). Thermocouple probes (ModelBat-12 from Physitemp Instruments Inc. of Clifton, N.J.) were placed inthe subcutaneous tissue and below the dura and measurements wererecorded at various power densities. The results of these measurementsare shown in Table 3:

TABLE 3 Animal Probe location Dose Exposure time Temperature increaseRat Subcutaneous 15 mW/cm² 4 minutes approximately 3° C. Rat Subdural 15mW/cm² 4 minutes approximately 1° C. Rat Subcutaneous 75 mW/cm² 4minutes approximately 7° C. Rat Subdural 75 mW/cm² 4 minutesapproximately 7° C. Rabbit Subcutaneous 7.5 mW/cm²  5 minutes less than0.5° C. Rabbit Subdural 7.5 mW/cm²  5 minutes less than 0.5° C. RabbitSubcutaneous 37.5 mW/cm²   5 minutes approximately 5.5° C. RabbitSubdural 37.5 mW/cm²   5 minutes less than 0.5° C.

There is minimal heating (e.g., less than 0.5° C.) in the subduralregion at four times the therapeutic energy density. The “heat sink”effect of living tissue that minimizes possible heating in the cortex issignificantly larger in humans than in rats or rabbits, due to thelarger heat sink and blood flow volume, which further limits theundesirable effects of heating in the treated region of the brain.Therefore, in certain embodiments described herein, a therapeutic dosageof energy is delivered to the target area of the brain withoutundesirable heating of the dura.

Treatment of Heat Stroke

In certain embodiments, a method prevents heat stroke in a subject. Theterm “preventing” in this context includes reducing the severity of alater heat stroke in a subject that has undergone treatment, reducingthe incidence of heat stroke in individuals who have undergonetreatment, as well as reducing the likelihood of onset heat stroke in asubject that has undergone treatment. The method includes deliveringlight energy having a wavelength in the visible to near-infraredwavelength range through the skull to at least one area of the brain ofa subject, wherein the wavelength, power density and amount of the lightenergy delivered are sufficient to prevent, reduce the severity, orreduce the incidence of heat stroke in the subject.

EXAMPLES Example 1: Effect of Phototherapy on ATP Production in Neurons

An in vitro experiment was done to demonstrate one effect ofphototherapy on neurons, namely the effect on ATP production. NormalHuman Neural Progenitor (NHNP) cells were obtained cryopreserved throughClonetics of Baltimore, Md., catalog # CC-2599. The NHNP cells werethawed and cultured on polyethyleneimine (PEI) with reagents providedwith the cells, following the manufacturers' instructions. The cellswere plated into 96 well plates (black plastic with clear bottoms,Becton Dickinson of Franklin Lakes, N.J.) as spheroids and allowed todifferentiate into mature neurons over a period of two weeks.

A Photo Dosing Assembly (PDA) was used to provide precisely metereddoses of laser light to the NHNP cells in the 96 well plates. The PDAincluded a Nikon Diaphot inverted microscope (Nikon of Melville, N.Y.)with a LUDL motorized x, y, z stage (Ludl Electronic Products ofHawthorne, N.Y.). An 808 nanometer laser was routed into the rearepi-fluorescent port on the microscope using a custom designed adapterand a fiber optic cable. Diffusing lenses were mounted in the path ofthe beam to create a “speckled” pattern, which was intended to mimic invivo conditions after a laser beam passed through human skin. The beamdiverged to a 25 millimeter diameter circle when it reached the bottomof the 96 well plates. This dimension was chosen so that a cluster offour adjacent wells could be lased at the same time. Cells were platedin a pattern such that a total of 12 clusters could be lased per 96 wellplate. Stage positioning was controlled by a Silicon Graphicsworkstation and laser timing was performed by hand using a digitaltimer. The measured power density passing through the plate for the NHNPcells was 50 mW/cm².

Two independent assays were used to measure the effects of 808 nanometerlaser light on the NHNP cells. The first was the CellTiter-GloLuminescent Cell Viability Assay (Promega of Madison, Wis.). This assaygenerates a “glow-type” luminescent signal produced by a luciferasereaction with cellular ATP. The CellTiter-Glo reagent is added in anamount equal to the volume of media in the well and results in celllysis followed by a sustained luminescent reaction that was measuredusing a Reporter luminometer (Turner Biosystems of Sunnyvale, Calif.).Amounts of ATP present in the NHNP cells were quantified in RelativeLuminescent Units (RLUs) by the luminometer.

The second assay used was the alamarBlue assay (Biosource of Camarillo,Calif.). The internal environment of a proliferating cell is morereduced than that of a non-proliferating cell. Specifically, the ratiosof NADPH/NADP, FADH/FAD, FMNH/FMN and NADH/NAD, increase duringproliferation. In several embodiments, laser irradiation may have aneffect on one or more of these ratios. Compounds such as alamarBlue arereduced by these metabolic intermediates and can be used to monitorcellular states. The oxidization of alamarBlue is accompanied by ameasurable shift in color. In its unoxidized state, alamarBlue appearsblue; when oxidized, the color changes to red. To quantify this shift, a340PC microplate reading spectrophotometer (Molecular Devices ofSunnyvale, Calif.) was used to measure the absorbance of a wellcontaining NHNP cells, media and alamarBlue diluted 10% v/v. Theabsorbance of each well was measured at 570 nanometers and 600nanometers and the percent reduction of alamarBlue was calculated usingan equation provided by the manufacturer.

The two metrics described above, (RLUs and % Reduction) were then usedto compare NHNP culture wells that had been lased with 50 mW/cm² at awavelength of 808 nanometers. For the CellTiter-Glo assay, 20 wells werelased for 1 second and compared to an unlased control group of 20 wells.The CellTiter-Glo reagent was added 10 minutes after lasing completedand the plate was read after the cells had lysed and the luciferasereaction had stabilized. The average RLUs measured for the control wellswas 3808+/−3394 while the laser group showed a two-fold increase in ATPcontent to 7513+/−6109. The standard deviations were somewhat high dueto the relatively small number of NHNP cells in the wells (approximately100 per well from visual observation), but a student's unpaired t-testwas performed on the data with a resulting p-value of 0.02 indicatingthat the two-fold change is statistically significant.

The alamarBlue assay was performed with a higher cell density and alasing time of 5 seconds. The plating density (calculated to be between7,500-26,000 cells per well based on the certificate of analysisprovided by the manufacturer) was difficult to determine since some ofthe cells had remained in the spheroids and had not completelydifferentiated. Because plating conditions were identical, wells fromthe same plate were compared. The alamarBlue was added immediately afterlasing and the absorbance was measured 9.5 hours later. The averagemeasured values for percent reduction were 22%+/−7.3% for the 8 lasedwells and 12.4%+/−5.9% for the 3 unlased control wells (p-value=0.076).Thus, in several embodiments, the laser treatment results in a positivemetabolic effect in the lased cells.

Several embodiments of the invention increase ATP concentrations, andare particularly advantageous because increases in cellular ATPconcentration and a more reduced state within the cell are both relatedto cellular metabolism and are considered to be indications that thecell is viable and healthy. In some embodiments, positive effects oflaser irradiation on cellular metabolism in in-vitro neuronal cellcultures are achieved.

Example 2: Transcranial Laser Therapy for Treatment of Stroke

In a second set of experiments, transcranial laser therapy for strokewas investigated using a low-energy infrared laser to treat behavioraldeficits in a rabbit small clot embolic stroke model (RSCEM). Thisexample is described in more detail by P. A. Lapchak et al.,“Transcranial Infrared Laser Therapy Improves Clinical Rating ScoresAfter Embolic Strokes in Rabbits,” Stroke, Vol. 35, pp. 1985-1988(2004), which is incorporated in its entirety by reference herein.

RSCEM was produced by injection of blood clots into the cerebralvasculature of anesthetized male New Zealand White rabbits, resulting inischemia-induced behavioral deficits that can be measured quantitativelywith a dichotomous rating scale. In the absence of treatment, smallnumbers of microclots caused no grossly apparent neurologic dysfunctionwhile large numbers of microclots invariably caused encephalopathy ordeath. Behaviorally normal rabbits did not have any signs of impairment,whereas behaviorally abnormal rabbits had loss of balance, head leans,circling, seizure-type activity, or limb paralysis.

For laser treatment, a laser probe was placed in direct contact with theskin. The laser probe comprised a low-energy laser (wavelength of 808±5nanometers) fitted with an OZ Optics Ltd. fiber-optic cable and a laserprobe with a diameter of approximately 2 centimeters. Instrument designstudies showed that these specifications would allow for laserpenetration of the rabbit skull and brain to a depth of 2.5 to 3centimeters, and that the laser beam would encompass the majority of thebrain if placed on the skin surface posterior to bregma on the midline.Although the surface skin temperature below the probe was elevated by upto 3° C., the focal brain temperature directly under the laser probe wasincreased by 0.8° C. to 1.8° C. during the 10-minute laser treatmentusing the 25 mW/cm² energy setting. Focal brain temperature returned tonormal within 60 minutes of laser treatment.

The quantitative relationship between clot dose and behavioral orneurological deficits was evaluated using logistic (S-shaped) curvesfitted by computer to the quantal dose-response data. These parametersare measures of the amount of microclots (in mg) that producedneurologic dysfunction in 50% of a group of animals (P₅₀). A separatecurve was generated for each treatment condition, with a statisticallysignificant increase in the P₅₀ value compared with control beingindicative of a behavioral improvement. The data were analyzed using thet test, which included the Bonferroni correction when appropriate.

To determine if laser treatment altered physiological variables, 14rabbits were randomly divided into 2 groups, a control group and alaser-treated group (25 mW/cm² for 10 minutes). Blood glucose levelswere measured for all embolized rabbits using a Bayer Elite XL 3901BGlucometer, and body temperature was measured using a Braun ThermoscanType 6013 digital thermometer. Within 60 minutes of embolization, therewas an increase in blood glucose levels in both the control group andthe laser-treated group that was maintained for the 2 hourspost-embolization observation time. Blood glucose levels returned tocontrol levels by 24 hours, regardless of the extent of stroke-inducedbehavioral deficits. Laser treatment did not significantly affectglucose levels at any time. Neither embolization nor laser treatmentsignificantly affected body temperature in either group of rabbits.

FIG. 43A is a graph for the percentage of the population which waseither abnormal or dead as a function of the clot weight in milligramsfor laser treatment of 7.5 mW/cm² for a treatment duration of 2 minutes.As shown by FIG. 43A, the control curve (dotted line) has a P₅₀ value of0.97±0.19 mg (n=23). Such laser treatment initiated 3 hours after thestroke significantly improved behavioral performance, with the P₅₀ valueincreased to 2.21±0.54 mg (n=28,*P=0/05) (solid line). The effect wasdurable and was measurable 3 weeks after embolization. However, the samesetting did not improve behavior if there was a long delay (24 hours)after embolization (dashed line) (P₅₀=1.23±0.15 mg, n=32).

In several embodiments, as discussed above, LLLT is advantageous ifstarted soon after an injury (e.g., traumatic injury or onset ofstroke). In several embodiments, treatment is administered as soon aspossible after an injury. In some embodiments, a plurality of additionaltreatments are made after an initial treatment. In some embodiments,incorporation of a delay before, during, or after therapy (e.g., priorto a second treatment) is beneficial. For example, in some embodiments,improved therapeutic effects are obtained if there a purposeful delaybetween an injury and the inception of treatment. In some embodiments,outcomes are improved if an immediate treatment is made, followed by adelay prior to a second treatment. In several embodiments, the delay ison the order of hours, for example, between about 1 and 8 hours after aninjury, including 1-2 hours, 2-4 hours, 3-5 hours, 4-6 hours, 5-7 hours,and 7-8 hours after injury. In some embodiments, treatment is delayedbetween about 6 to 24 hours after injury (including 6-8 hours, 8-10hours, 10-12 hours, 12-14 hours, 14-18 hours, 18-20 hours, 20-22 hoursand 22-24 hours, and overlapping ranges thereof). In some embodiments,treatment is delayed from 24-48 hours. In several embodiments the delaysare tailored to the response characteristics of an individual patient(e.g., tailored therapy).

FIG. 43B is a graph for the percentage of the population which waseither abnormal or dead as a function of the clot weight in milligramsfor laser treatment of 25 mW/cm2 for a treatment duration of 10 minutes.As shown by FIG. 43B, the control curve (dotted line) has a P₅₀ value of1.10±0.17 mg (n=27). Such laser treatment initiated 1 (dashed line) or 6(solid line) hours after embolization also significantly increasedbehavioral performance, with the P₅₀ value increased to 2.02±0.46 mg(n=18,*P<0.05) and 2.98±0.65 mg (n=26,*P<0.05), respectively.

FIG. 44 is a graph showing the therapeutic window for laser-inducedbehavioral improvements after small-clot embolic strokes in rabbits.Results are shown as clinical rating score P₅₀ (mg clot) given asmean±SEM for the number of rabbits per time point (number in brackets)for laser treatment initiated 1, 3, 6, or 24 hours after embolization asshown on the x-axis. The horizontal line represents the mean of thecontrol P₅₀ values (*P<0.05).

The results in the RSCEM showed that laser treatment significantlyimproved behavioral rating scores after embolic strokes in rabbitswithout affecting body temperature and blood glucose levels. Inaddition, laser treatment was effective when initiated up to 6 hoursafter strokes, which is later than any other previously effective singletherapy in the same preclinical stroke model. Moreover, the effect wasdurable and was measurable up to 21 days after embolization. In severalembodiments, LLLT provides a viable therapeutic regime for the treatmentof stroke (or other brain/neural injuries) that results in long-lastingimprovements (e.g., recoveries) in neurological function. The magnitudesof laser-induced improvement in rabbits are similar to previously testedthrombolytics (alteplase, tenecteplase, and microplasmin) andneuroprotective compounds (NXY-059), which are undergoing clinicaldevelopment. Thus, several embodiments are particularly advantageous asan alternative or supplement to pharmacological therapies. In someembodiments, LLLT works synergistically with pharmacological therapies.For example, in one embodiment, LLLT enhances the therapeutic effect ofa given dose of a drug. In another embodiment, LLLT reduces the dose ofa drug needed to achieve a comparable effect.

Example 3: LLLT in Combination with Neural Stem Cells to TreatNeurodegeneration

Given the propensity of neurological disorders or acute neural injury toinduce a loss of function of one or more neural cells, and thedisclosure herein regarding the positive effects of LLLT on cells, inparticular neural stem cells, the present experiment will be directed tothe combination of LLLT and neural stem cell therapy. Cultured healthyneurons will be used to investigate the effects of LLLT and neural stemcell therapy in combination.

Multiple populations of healthy neurons will be cultured under standardsterile tissue culture conditions. A population of healthy neurons willbe exposed to rotenone, a chemical stimulus that induces a loss offunction in the neurons and is an accepted model for Parkinson'sdisease, while another population will be maintained under originalconditions as a control. The rotenone exposed neurons will exhibit lossof one or more functional endpoints as compared to the control neurons(e.g., diminished action potential duration, diminished neurotransmitterrelease).

Multiple populations of neural progenitor (stem) cells will also becultured. A population of neural progenitors will be exposed to LLLTaccording to parameters disclosed herein. Another population will bemaintained unexposed as a control. In one embodiment, LLLT exposure willenhance the viability of the neural progenitors. In one embodiment, theLLLT exposure will enhance the ability of the neural progenitor cells todifferentiate based on the environmental cues (e.g., growth factors ordifferentiation-inducing agents in vitro or in vivo).

LLLT-exposed and unexposed neural progenitor cells will be co-culturedwith healthy and rotenone-treated neurons. In several embodiments, theenhanced differentiation of the LLLT-treated cells will increase thenumber of neural progenitor cells that differentiate into neurons of thesame type as in the co-culture. As a result, in one embodiment,rotenone-treated neurons co-cultured with LLLT-treated neural progenitorcells will exhibit recovery from the rotenone-induced loss of function.The recovery will be enhanced as compared to the co-culture employingnon-LLLT treated neural progenitors.

In vivo experiments will also be performed. Control and rotenone-treatedrats will be used. Rotenone-treated rats will exhibit loss of functionas assessed by clinical endpoints for evaluation of Parkinson's disease(e.g., motor control and behavioral tests). LLLT-treated or controlneural progenitor cells are stereotactically administered to thesubstantia nigra of control and rotenone-treated rats. In severalembodiments, rotenone-treated rats receiving LLLT-treated neuralprogenitor cells will exhibit increased motor control and improvedclinical assessment scores. In one embodiment, immunohistochemicalanalysis of brains of rotenone-treated rats receiving LLLT-treatedneural progenitor cells will reveal a larger percentage of LLLT-treatedneural progenitor cells differentiated into functional neurons, therebyreplacing the rotenone-damaged neurons, and accounting for the increasedefficacy of cell therapy.

In one embodiment, similar results will be obtained when LLLT-treatedprogenitors are administered to Alzheimer's rats (e.g., Samaritan FABRat Model).

Example 4: LLLT in Combination with Neural Stem Cells to Treat AcuteNeural Injury

In an additional experiment, rabbits will be subjected to traumaticbrain injury (TBI) according to an art-recognized model (e.g.,controlled mass weight drop) and will be treated with the combination ofstem cells and LLLT. A group of healthy rabbits will be used as acontrol. Another group of TBI rabbits that do not receive cells willserve as another control. TBI rabbits will exhibit neural damages asassessed by standard neurological severity scales. Neural progenitorcells will be cultured and treated as described above. LLLT-treated anduntreated neural stem cells will be administered to healthy and TBIrabbits. In several embodiments, TBI rabbits receiving LLLT-treatedneural stem cells will exhibit improved neurological scores as comparedto TBI rabbits which did not receive cells. Further, TBI rabbitsreceiving LLLT-treated neural stem cells will exhibit enhanced recoveryof function as compared to TBI rabbits receiving untreated stem cells.In one embodiment, immunohistochemical analysis of brains of TBI rabbitsreceiving LLLT-treated neural stem cells will reveal that a largerpercentage of LLLT-treated neural stem cells differentiated intofunctional neurons, thereby replacing the TBI-damaged neurons, andaccounting for the increased efficacy of cell therapy.

In one embodiment, similar results are achieved when LLLT-treated neuralprogenitor cells are administered to rabbits receiving surgicallyinduced ischemic stroke.

Example 5: LLLT Treatment of Progenitor Cells Enhances the TherapeuticPotential of the Cells

As discussed above, increasing the therapeutic efficacy of cell therapyis desirable. In an additional experiment, LLLT will be administered tocultured neural stem cells according to parameters disclosed herein.Another population of neural stem cells will serve as an untreatedcontrol. In several embodiments, LLLT treatment will increase theresponse of the stem cells to pro-differentiation factors (e.g., growthfactors) as compared to the control cells. In several embodiments, LLLTtreatment will also improve the ability of the stem cells to survive inadverse growth conditions (e.g., hypoxia, lack of essential growthnutrient). In some embodiments, LLLT treated cells will respond tochemoattractants to a greater degree than control cells, indicatingenhanced migratory capacity. In some embodiments, injection ofLLLT-treated stem cells into a host tissue (e.g. muscle, brain) willyield a higher degree of engraftment as compared to untreated cells.Improvement in each of these parameters will indicate that stem cellshave increased therapeutic potential.

Various embodiments have been described above. Although this inventionhas been described with reference to these specific embodiments, thedescriptions are intended to be illustrative of the invention and arenot intended to be limiting. Various modifications and applications mayoccur to those skilled in the art without departing from the true spiritand scope of the invention.

What is claimed is:
 1. A method for enhancing the suitability of neuralstem cells for use in neural cell therapy comprising: obtaining apopulation of neural stem cells; providing a low level light therapy(LLLT) device, wherein the LLLT device has a light emitting surface thatemits light energy; delivering a first light energy from the LLLT deviceto the neural stem cells in vitro; after delivering the first lightenergy, administering the neural stem cells to an impaired tissue; andafter administering the neural stem cells to the impaired tissue,delivering a second light energy from the LLLT device to the neural stemcells in vivo, wherein at least one of the first light energy and thesecond light energy has a time averaged irradiance at or within onecentimeter of the stem cells of 0.01 mW/cm² to 1 W/cm², and wherein atleast one of the first light energy and the second light energyincreases one or more of the migration and engraftment of the stemcells.
 2. The method of claim 1, wherein at least one of the first lightenergy and the second light energy has a wavelength between 630 nm and904 nm.
 3. The method of claim 1, wherein the stem cells are derivedfrom the group of stem cell sources consisting of adult stem cells,embryonic stem cells, placenta-derived stem cells, bone marrow-derivedstem cells, mesenchymal stem cells, adipose stem cells, and inducedpluripotent stem cells.
 4. The method of claim 1, wherein the stem cellsare for use in cell therapy to treat a neurological disease or injury.5. The method of claim 4, wherein the neurological disease or injury isselected from the group consisting of Parkinson's disease, Alzheimer'sdisease, Huntington's disease, dopaminergic impairment, depression,stroke, head trauma, neurodegeneration, and dementia.
 6. The method ofclaim 1, wherein at least one of the first light energy and the secondlight energy has a time averaged irradiance at or within one centimeterof the stem cells of 20 mW/cm² to 60 mW/cm².
 7. The method of claim 1,wherein at least one of the first light energy and the second lightenergy has a time averaged energy density at or within one centimeter ofthe stem cells of 0.5 J/cm² to 5 J/cm².
 8. The method of claim 1,wherein at least one of the first light energy and the second lightenergy is pulsed.